Non-polar media for polynucleotide separations

ABSTRACT

Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.

CROSS REFERENCE TO RELATED CO-PENDING APPLICATIONS

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 09/058,337 filed Apr. 10, 1998 which is hereby incorporated byreference in its entirety. This application is a regular U.S. patentapplication under 35 U.S.C. §111(a) and 37 C.F.R. §1.53(b) and claimspriority from the following copending, commonly assigned provisionalapplications, each filed under 35 U.S.C. §111(b):

[0002] Ser. No. 60/069,313, filed Dec. 5, 1997

[0003] Ser. No. 60/077,998, filed Mar. 13, 1998

[0004] Ser. No. 60/089,606, filed Jun. 17, 1998

[0005] Ser. No. 60/103,313, filed Oct. 6, 1998.

FIELD OF THE INVENTION

[0006] The present invention is directed to the separation ofpolynucleotides using a separation medium having non-polar surfaces,such as the surfaces of nonporous beads or surfaces of interstitialspaces within a molded monolith (e.g., a derivatized silica monolith),which surfaces are substantially free from contamination withmultivalent cations. More specifically, the invention is directed to thechromatographic separation of both single stranded and double strandedpolynucleotides by chromatography using a nonporous separation medium,where the medium is either organic or inorganic material which is coatedwith a polymer, or non-polar substituted polymer, and/or which hassubstantially all surface substrate groups substituted with a-non-polarhydrocarbon or non-ionic substituted hydrocarbon.

BACKGROUND OF THE INVENTION

[0007] Separations of polynucleotides such as DNA have beentraditionally performed using slab gel electrophoresis or capillaryelectrophoresis. However, liquid chromatographic separations ofpolynucleotides are becoming more important because of the ability toautomate the analysis and to collect fractions after they have beenseparated. Therefore, columns for polynucleotide separation by liquidchromatography (LC) are becoming more important.

[0008] Silica-based columns are by far the most common LC columns. Ofthese, reverse phase silica-based columns are preferred because theyhave high separation efficiencies, are mechanically stable, and avariety of functional groups can be easily attached for a variety ofcolumn selectivities.

[0009] Although silica-based reverse phase column materials haveperformed adequately for separating single stranded DNA, these materialshave not performed well for separating double stranded DNA. The peaksfrom double stranded DNA separations using silica-based materials arebadly shaped or broad, or the double stranded DNA may not even elute.Separations can take up to several hours, or the resolution, peaksymmetry, and sensitivity of the separation are poor.

[0010] High quality materials for DNA separations have been based onpolymeric substrates, as disclosed in U.S. Pat. No. 5,585,236 to Bonn(1966). There exists a need for silica-based column packing material andother materials that are suitable for separation of double stranded DNA.

SUMMARY OF THE INVENTION

[0011] Accordingly, one object of the present invention is to provide achromatographic method for separating polynucleotides with improvedseparation and efficiency. Another object is to provide improvednon-polar separation media for the separation of polynucleotides.

[0012] These and other objects of the invention, which will becomeapparent from reading the following specification, have been achieved bythe method of the present invention in which polynucleotides areseparated using a nonporous separation medium such as beads or a moldedmonolith (e.g., a silica gel monolith), where the medium compriseseither organic or inorganic material which is coated with a polymer, ornon-polar substituted polymer, and/or which has substantially allsurface substrate groups substituted with a non-polar hydrocarbon ornon-ionic substituted hydrocarbon.

[0013] In one aspect, the invention is a method for separating a mixtureof polynucleotides comprising applying a mixture of polynucleotideshaving up to 1500 base pairs to a separation medium, the separationsurfaces of the medium coated with a hydrocarbon or non-polarhydrocarbon substituted polymer, or having substantially all polargroups reacted with a non-polar hydrocarbon or substituted hydrocarbongroup, wherein said surfaces are non-polar; and eluting thepolynucleotides. The separation medium can be enclosed in a column.Examples of non-polar surfaces include the surfaces of beads such asnonporous particles and the surfaces of intersitital spaces within amonolith (e.g., a silica gel monolith), which surfaces are coated with ahydrocarbon or non-polar substituted polymer or having substantially allsurface substrate groups reacted with a non-polar hydrocarbon orsubstituted hydrocarbon group. In the preferred embodiment, precautionsare taken during the production of the medium so that it issubstantially free of multivalent cation contaminants and the medium istreated, for example by an acid wash treatment and/or treatment withmultivalent cation binding agent, to substantially remove any residualsurface metal contaminants. The preferred separation medium ischaracterized by having a DNA Separation Factor (defined hereinbelow) ofat least 0.05. The preferred medium is characterized by having aMutation Separation Factor (as defined hereinbelow) of at least 0.1. Ina preferred embodiment, the separation is made by Matched IonPolynucleotide Chromatography (MIPC, as defined hereinbelow). Theelution step preferably uses a mobile phase containing a counterionagent and a water-soluble organic solvent. Examples of a suitableorganic solvent include alcohol, nitrile, dimethylformamide,tetrahydrofuran, ester, ether, and mixtures of one or more thereof,e.g., methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran, ethylacetate, acetonitrile. The most preferred organic solvent isacetonitrile. The counterion agent is preferably selected from the groupconsisting of lower primary amine, lower secondary amine, lower tertiaryamine, lower trialkyammonium salt, quaternary ammonium salt, andmixtures of one or more thereof. Non-limiting examples of counterionagents include octylammonium acetate, octyldimethylammonium acetate,decylammonium acetate, octadecylammonium acetate, pyridiniumammoniumacetate, cyclohexylammonium acetate, diethylammonium acetate,propylethylammonium acetate, propyldiethylammonium acetate,butylethylammonium acetate, methylhexylammonium acetate,tetramethylammonium acetate, tetraethylammonium acetate,tetrapropylammonium acetate, tetrabutylammonium acetate,dimethydiethylammonium acetate, triethylammonium acetate,tripropylammonium acetate, tributylammonium acetate, and mixtures of anyone or more of the above. The counterion agent includes an anion, e.g.,acetate, carbonate, bicarbonate, phosphate, sulfate, nitrate,propionate, formate, chloride, perchlorate, or bromide. The mostpreferred counterion agent is triethylammonium acetate ortriethylammonium hexafluoroisopropyl alcohol.

[0014] One embodiment of the invention provides a method for separatinga mixture of polynucleotides, comprising applying a mixture ofpolynucleotides having up to 1500 base pairs to separation beads havingnon-polar surfaces, and eluting said mixture of polynucleotides. In aparticular embodiment of the separation medium, the invention provides amethod for separating a mixture of polynucleotides comprising applying amixture of polynucleotides having up to 1500 base pairs through aseparation column containing beads which are substantially free fromcontamination with multivalent cations and having an average diameter of0.5 to 100 microns, and eluting the mixture of polynucleotides. In oneembodiment, the beads comprise nonporous particles coated with ahydrocarbon or non-polar substituted polymer or having substantially allsurface substrate groups reacted with a non-polar hydrocarbon orsubstituted hydrocarbon group. The beads preferably have an averagediameter of about 1-5 microns. In the preferred embodiment, precautionsare taken during the production of the beads so that they aresubstantially free of multivalent cation contaminants and the beads aretreated, for example, by an acid wash treatment and/or treatment withmultivalent cation binding agent, to remove any residual surface metalcontaminants. The beads of the invention are characterized by having aDNA Separation Factor of at least 0.05. In a preferred embodiment, thebeads are characterized by having a DNA Separation Factor of at least0.5. Also in a preferred embodiment, the beads are characterized byhaving a Mutation Separation Factor of at least 0.1. In one embodiment,the beads are used in a capillary column to separate a mixture ofpolynucleotides by capillary electrochromatography. In otherembodiments, the beads are used to separate the mixture by thin-layerchromatography or by high-speed thin-layer chromatography. Theseparation is preferably by MIPC. The beads preferably have an averagediameter of about 1-5 microns. The nonporous particle is preferablyselected from silica, silica carbide, silica nitrite, titanium oxide,aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides suchas cellulose, and diatomaceous earth, or any of these materials thathave been modified to be nonporous. The nonporous particle is mostpreferably silica, which preferably is substantially free fromunderivatized silanol groups. The particles can be prepared bynon-covalently bonded coatings, covalently bonded coatings, or reactionof the silanol groups with hydrocarbon groups.

[0015] The nonporous particle can be coated with a polymer. The polymeris preferably selected from polystyrenes, polymethacrylates,polyethylenes, polyurethanes, polypropylenes, polyamides, cellulose,polydimethyl siloxane, and polydialkyl siloxane. The polymer isoptionally unsubstituted or substituted with hydrocarbon groups or othergroups having nonionic substituents. The polymer can be optionallysubstituted with hydrocarbon groups having from 1 to 1,000,000 carbons,the hydrocarbon groups optionally being alkyl groups with from 1 to 100carbons and preferably from 1 to 24 carbons. Hydrocarbon groups from 24to 1,000,000 are described herein as hydrocarbon polymers and have theconstituency of hydrocarbon groups as defined herein.

[0016] The reaction of organosilanols (e.g. HO—Si—R₃) or alkoxy- (e.g.,RO—Si—R₃) silanes with silica supports without polymerization can alsoproduce good packings. The method produces a dense monolayer offunctional groups of alkyl or alkylsubstituted, ester, cyano, and othernonionic groups. The use of monofunctional dimethyl silanes(X—Si(CH₃)₂—R) provides a homogeneous organic coating with a minimum ofresidual Si—OH groups. Monochlorosilane reagents are preferred, if therequired organic functionality can be prepared. These reactions arereproducible and provide high quality packing materials. Unreacted,accessible silanols can be left after the initial reaction. Thenonporous particle is preferably endcapped with a tri(loweralkyl)chlorosilane (preferably a trimethylchlorosilane) to blockresidual reactive silanol sites following the coating or hydrocarbonsubstitution. Alternatively, all of the silanol sites can be reactedwith an excess of the endcapping reagent to extinguish all reactivesilanol groups. Endcapping of the nonporous particle can be effected byreaction of the nonporous particle with the corresponding hydrocarbonsubstituted halosilane, such as trialkyl chlorosilane (eg. trimethylchlorosilane) or by reaction with the corresponding hydrocarbonsubstituted disilazane, such as dichloro-tetraalkyl-disilazane (eg.dichloro-tetramethyl-disilazane).

[0017] The method of the present invention can be used to separatedouble stranded polynucleotides having up to about 1500 to 2000 basepairs. In many cases, the method is, used to separate polynucleotideshaving up to 600 bases or base pairs, or which have up to 5 to 80 basesor base pairs.

[0018] The method is performed at a temperature within the range of 20°C. to 90° C. The flow-rate of the mobile phase is preferably adjusted toyield a back-pressure not greater than 10,000 psi. The method alsopreferably employs an organic solvent and more preferably an organicsolvent that is water soluble. The solvent is preferably selected fromthe group consisting of alcohols, nitrites, dimethylformamide, esters,and ethers. The method also preferably employs a counterion agentselected from trialkylamine acetate, trialkylamine carbonate, andtrialkylamine phosphate. The most preferred counterion agent istriethylammonium acetate or triethylammonium hexafluoroisopropylalcohol.

[0019] In addition to the beads or other medium being substantiallymetal-free, Applicants have also found, that to achieve optimum peakseparation, the inner surfaces of the column (or other container) andall process solutions held within the separation system or flowingthrough the system are preferably substantially free of multivalentcation contaminants. The method preferably comprises supplying andfeeding solutions entering the separation column with components havingprocess solution-contacting surfaces which contact process solutionsheld therein or flowing therethrough. The process solution-contactingsurfaces are material which does not release multivalent cations intoaqueous solutions held therein or flowing therethrough, so that thecolumn and its contents are protected from multivalent cationcontamination The process solution-contacting surfaces are preferablymaterial selected from the group consisting of titanium, coatedstainless steel, passivated stainless steel, and organic polymer.Multivalent cations in mobile phase solutions and sample solutionsentering the column are also preferably removed by contacting thesolutions with multivalent cation capture resin before the solutionsenter the column so as to protect the separation medium from multivalentcation contamination. The multivalent capture resin is selected fromcation exchange resin and chelating resin. The column and processsolutions held therein or flowing therethrough are preferablysubstantially free of multivalent cation contaminants. Thepolynucleotides are separated by Matched Ion PolynucleotideChromatography.

[0020] Also disclosed herein is a method for separating a mixture ofpolynucleotides, comprising flowing a mixture of polynucleotides havingup to 1500 base pairs through a separation column containing beadshaving an average diameter of 0.5 to 100 microns, and separating themixture of polynucleotides by Matched Ion Polynucleotide Chromatography.The beads comprise nonporous particles coated with a polymer or havingsubstantially all surface substrate groups reacted and/or endcapped witha non-polar hydrocarbon or substituted hydrocarbon group. The beads arecharacterized by having a DNA Separation Factor of at least 0.05.

[0021] Also disclosed herein is a bead comprising a nonporous particlecoated with a polymer. The bead has an average diameter of 0.5 to 100microns and is characterized by having a DNA Separation Factor of atleast 0.05. In a preferred embodiment, the bead is characterized byhaving a DNA Separation Factor of at least 0.5. The preferred bead ischaracterized by having a Mutation Separation Factor of at least 0.1.The bead preferably has a diameter of about 1-5 microns. The nonporousparticle is preferably selected from silica, silica carbide, silicanitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon,insoluble polysaccharides such as cellulose, and diatomaceous earth, orany of these materials that have been modified to be nonporous. Thenonporous particle is most preferably silica, which preferably hasminimum silanol groups. The polymer is preferably selected frompolystyrene, polymethacrylate, polyethylene, polyurethane,polypropylene, polyamide, cellulose, polydimethyl siloxane, andpolydialkyl siloxane, and is preferably unsubstituted, alkylated, oralkyl or aryl substituted, or alkylated with a substituted alkyl groupmethyl-substituted, or ethyl-substituted. The polymer can be alkylatedwith alkyl groups having 1-22 carbon atoms, preferably, 8-18 carbonatoms.

[0022] Also disclosed herein is a bead comprising a nonporous particlehaving substantially all surface substrate groups reacted with ahydrocarbon group and then endcapped with a non-polar hydrocarbon orsubstituted hydrocarbon group, preferably a tri(lower alkyl)chlorosilaneor tetra(lower alkyl)dichlorodisilazane. The bead has an averagediameter of 0.5 to 100 microns and is characterized by having a DNASeparation Factor of at least 0.05. The bead preferably has a diameterof about 1-5 microns.

[0023] The nonporous particle is preferably selected from silica, silicacarbide, silica nitrite, titanium oxide, aluminum oxide, zirconiumoxide, carbon, insoluble polysaccharides such as cellulose, anddiatomaceous earth, or any of these materials that have been modified tobe nonporous. The nonporous particle is most preferably silica, whichpreferably has minimum silanol groups. Endcapping of the nonporousparticle can be effected by reaction of the nonporous particle withtrimethyl chlorosilane or dichloro-tetraisopropyl-disilazane.

[0024] In a still further aspect, the invention is a method forseparating a mixture of polynucleotides comprising applying a mixture ofpolynucleotides having up to 1500 base pairs to a monolith havingnon-polar separation surfaces, and eluting the polynucleotides. Themonolith can be enclosed in a column or other containment system, suchas a cartridge. In a preferred embodiment, the monolith is a silica gelmonolith. The non-polar separation surfaces include the surfaces ofintersitital spaces within the monolith, which surfaces are coated witha hydrocarbon or non-polar substituted polymer or having substantiallyall surface substrate groups reacted with a non-polar hydrocarbon orsubstituted hydrocarbon group. An example of a suitable monolith is onewhich is polyfunctionally derivatized with octadecylsilyl groups. In thepreferred embodiment, precautions are taken during the production of themonolith so that it is substantially free of multivalent cationcontaminants and the monolith is treated, for example by an acid washtreatment and/or treatment with multivalent cation binding agent, tosubstantially remove any residual surface metal contaminants. Thepreferred monolith is characterized by having a DNA Separation Factor ofat least 0.05. The preferred monolith is characterized by having aMutation Separation Factor of at least 0.1. In a preferred embodiment,the separation is made by Matched Ion Polynucleotide Chromatography. Theelution step preferably uses a mobile phase containing a counterionagent and a water-soluble organic solvent. Examples of a suitableorganic solvent include alcohol, nitrile, dimethylformamide,tetrahydrofuran, ester, ether, and mixtures of one or more thereof,e.g., methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran, ethylacetate, acetonitrile. The most preferred organic solvent isacetonitrile. The counterion agent is preferably selected from the groupconsisting of lower primary amine, lower secondary amine, lower tertiaryamine, lower trialkyammonium salt, quaternary ammonium salt, andmixtures of one or more thereof. Non-limiting examples of counterionagents include octylammonium acetate, octyldimethylammonium acetate,decylammonium acetate, octadecylammonium acetate, pyridiniumammoniumacetate, cyclohexylammonium acetate, diethylammonium acetate,propylethylammonium acetate, propyldiethylammonium acetate,butylethylammonium acetate, methylhexylammonium acetate,tetramethylammonium acetate, tetraethylammonium acetate,tetrapropylammonium acetate, tetrabutylammonium acetate,dimethydiethylammonium acetate, triethylammonium acetate,tripropylammonium acetate, tributylammonium acetate, and mixtures of anyone or more of the above. The counterion agent includes an anion, e.g.,acetate, carbonate, bicarbonate, phosphate, sulfate, nitrate,propionate, formate, chloride, perchlorate, or bromide. The mostpreferred counterion agent is triethylammonium acetate ortriethylammonium hexafluoroisopropyl alcohol.

[0025] In a yet further aspect, the invention provides a monolith havingnon-polar separation surfaces which are substantially free fromcontamination with multivalent cations. The monolith can be enclosed ina column or other containment system, such as a cartridge. The non-polarseparation surfaces include the surfaces of interstitial spaces withinthe monolith (e.g., a silica monolith), which surfaces are coated with ahydrocarbon or non-polar substituted polymer or having substantially allsurface substrate groups reacted with a non-polar hydrocarbon orsubstituted hydrocarbon group. An example of a suitable monolith is onewhich is derivatized with polyfunctionally derivatized octadecylsilylgroups. In the preferred embodiment, precautions are taken during theproduction of the monolith so that it is substantially free ofmultivalent cation contaminants and the monolith is treated, for exampleby an acid wash treatment and/or treatment with multivalent cationbinding agent, to remove any residual surface metal contaminants. Thepreferred monolith is characterized by having a DNA Separation Factor ofat least 0.05. The preferred monolith is characterized by having aMutation Separation Factor of at least 0.1.

[0026] In addition to the beads (or other media) themselves beingsubstantially metal-free, Applicants have also found that to achieveoptimum peak separation the inner surfaces of the separation column (orother container) and all process solutions held within the column orflowing through the column are preferably substantially free ofmultivalent cation contaminants. This can be achieved by supplying andfeeding solutions entering the separation column with components whichhave process solution-contacting surfaces made of material which doesnot release multivalent cations into the, process solutions held withinor flowing through the column, in order to protect the column frommultivalent cation contamination. The process solution-contactingsurfaces of the system components are preferably material selected fromthe group consisting of titanium, coated stainless steel, and organicpolymer. For additional protection, multivalent cations in mobile phasesolutions and sample solutions entering the column can be removed bycontacting these solutions with multivalent cation capture resin beforethe solutions enter the column to protect the separation medium frommultivalent cation contamination. The multivalent capture resin ispreferably cation exchange resin and/or chelating resin.

[0027] In another aspect, the present invention is a method for treatingthe non-polar surfaces of a medium used for separating polynculeotides,such as the surfaces of beads in a MIPC column or the surfaces ofinterstitial spaces in a monolith, in order to improve the resolution ofpolynucleotides, such as dsDNA, separated on said surfaces. Thistreatment includes contacting the surface with a solution containing amultivalent cation binding agent. In a preferred embodiment, thesolution has a temperature of about 50° C. to 90° C. An example of thistreatment includes flowing a solution containing a multivalent cationbinding agent through a MIPC column, wherein the solution has atemperature of about 50° C. to 90° C. The preferred temperature is about70° C. to 80° C. In a preferred embodiment, the multivalent cationbinding agent is a coordination compound, examples of which includewater-soluble chelating agents and crown ethers. Specific examplesinclude acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid,morin, rhodizonic acid, thionalide, thiourea, α-furildioxime, nioxime,salicylaldoxime, dimethylglyoxime, α-furildioxime, cupferron,α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone,diphenylcarbazone, eriochrome black T, PAN, SPADNS,glyoxal-bis(2-hydroxyanil), murexide, α-benzoinoxime, mandelic acid,anthranilic acid, ethylenediamine, glycine, triaminotriethylamine,thionalide, triethylenetetramine, ethylenediaminetetraacetic acid(EDTA), metalphthalein, arsenic acids, α,α′-bipyridine,4-hydroxybenzothiazole, 8-hydroxyquinaldine, 8-hydroxyquinoline,1,10-phenanthroline, picolinic acid, quinaldic acid, α,α′,α″-terpyridyl,9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol, salicylic acid,tiron, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole,rubeanic acid, oxalic acid, sodium diethyldithiocarbarbamate, and zincdibenzyldithiocarbamate. However, the most preferred chelating agent isEDTA. In this aspect of the invention, the solution preferably includesan organic solvent as exemplified by alcohol, nitrile,dimethylformamide, tetrahydrofuran, ester, ether, and mixtures thereof.Examples of suitable solvents include methanol, ethanol, 2-propanol,1-propanol, tetrahydrofuran, ethyl acetate, acetonitrile, and mixturesthereof. The most preferred organic solvent is acetonitrile. In oneembodiment, the solution can include a counterion agent such as lowerprimary, secondary and tertiary amines, and lower trialkyammonium salts,or quaternary ammonium salts. More specifically, the counterion agentcan be octylammonium acetate, octadimethylammonium acetate,decylammonium acetate, octadecylammonium acetate, pyridiniumammoniumacetate, cyclohexylammonium acetate, diethylammonium acetate,propylethylammonium acetate, propyldiethylammonium acetate,butylethylammonium acetate, methylhexylammonium acetate,tetramethylammonium acetate, tetraethylammonium acetate,tetrapropylammonium acetate, tetrabutylammonium acetate,dimethydiethylammonium acetate, triethylammonium acetate,tripropylammonium acetate, tributylammonium acetate, tetraethylammoniumacetate, tetrapropylammonium acetate, tetrabutylammonium acetate, andmixtures of any one or more of the above. The counterion agent includesan anion, e.g., acetate, carbonate, bicarbonate, phosphate, sulfate,nitrate, propionate, formate, chloride, perchlorate, and bromide.However, the most preferred counterion agent is triethylammoniumacetate.

[0028] In yet a further aspect, the invention provides a method forstoring a medium used for separating polynucleotides, e.g., the beads ofa MIPC column or a monolith, in order to improve the resolution ofdouble stranded DNA fragments separated using the medium. In the case ofa MIPC column, the preferred method includes flowing a solutioncontaining a multivalent cation binding agent through the column priorto storing the column. In a preferred embodiment, the multivalent cationbinding agent is a coordination compound, examples of which includewater-soluble chelating agents and crown ethers. Specific examplesinclude acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid,morin, rhodizonic acid, thionalide, thiourea, α-furildioxime, nioxime,salicylaldoxime, dimethylglyoxime, α-furildioxime, cupferron,α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone,diphenylcarbazone, eriochrome black T, PAN, SPADNS,glyoxal-bis(2-hydroxyanil), murexide, α-benzoinoxime, mandelic acid,anthranilic acid, ethylenediamine, glycine, triaminotriethylamine,thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids,α,α′-bipyridine, 4-hydroxybenzothiazole, 8-hydroxyquinaldine,8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid,α,α′,α″-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,salicylic acid, tiron, 4-chloro-1,2-dimercaptobenzene, dithiol,mercaptobenzothiazole, rubeanic acid, oxalic acid, sodiumdiethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate. However,the most preferred chelating agent is EDTA. In this aspect of theinvention, the solution preferably includes an organic solvent asexemplified by alcohols, nitrites, dimethylformamide, tetrahydrofuran,esters, and ethers. The most preferred organic solvent is acetonitrile.The solution can also include a counterion agent such as lower primary,secondary and tertiary amines, and lower trialkyammonium salts, orquaternary ammonium salts. More specifically, the counterion agent canbe octylammonium acetate, octadimethylammonium acetate, decylammoniumacetate, octadecylammonium acetate, pyridiniumammonium acetate,cyclohexylammonium acetate, diethylammonium acetate, propylethylammoniumacetate, propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, tributylammoniumacetate, tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, and mixtures of any one or more of theabove. The counterion agent includes an anion, e.g., acetate, carbonate,bicarbonate, phosphate, sulfate, nitrate, propionate, formate, chloride,perchlorate, and bromide. However, the most preferred counterion agentis triethylammonium acetate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a schematic representation of how the DNA SeparationFactor is applied to a separation.

[0030]FIG. 2 is a schematic drawing of a cross-section of arepresentation of a reverse phase bead with a silica core which has beenderivatized with a non-polar surface.

[0031]FIG. 3 is a schematic drawing of a cross-section of arepresentation of a reverse phase bead with a silica core and polymershielding.

[0032]FIG. 4 is a MIPC separation of pUC18 DNA-HaeIII digestionfragments on a column containing alkylated poly(styrene-divinylbenzene)beads. Peaks are labeled with the number of base pairs of the elutedfragment.

[0033]FIG. 5 is a MIPC separation of pUC18 DNA-HaeIII digestionfragments on a column containing nonporous 2.1 micron beads ofunderivatized poly(styrene-divinylbenzene).

[0034]FIG. 6 is a Van't Hoff plot of the retention factor 1/T[°K⁻¹] withalkylated poly(styrene-divinylbenzene) beads showing positive enthalpyusing acetonitrile as the solvent.

[0035]FIG. 7 is a Van't Hoff plot of the retention factor 1/T[°K⁻¹] withunderivatized poly(styrene-divinylbenzene) beads showing positiveenthalpy using acetonitrile as the solvent.

[0036]FIG. 8 is a Van't Hoff plot of the retention factor 1/T[°K⁻¹] withalkylated poly(styrene-divinylbenzene) beads showing negative enthalpyusing methanol as the solvent.

[0037]FIG. 9 is a separation using alkylated beads and acetonitrile assolvent.

[0038]FIG. 10 is a separation using alkylated beads and 50.0% methanolas the solvent.

[0039]FIG. 11 is a separation using alkylated beads and 25.0% ethanol asthe solvent.

[0040]FIG. 12 is a separation using alkylated beads and 25.0% vodka(Stolichnaya, 100 proof) as the solvent.

[0041]FIG. 13 is a separation using alkylated beads and 25.0% 1-propanolas the solvent.

[0042]FIG. 14 is a separation using alkylated beads and 25.0% 1-propanol as the solvent.

[0043]FIG. 15 is a separation using alkylated beads and 10.0% 2-propanolas the solvent.

[0044]FIG. 16 is a separation using alkylated beads and 10.0% 2-propanolas the solvent.

[0045]FIG. 17 is a separation using alkylated beads and 25.0% THF as thesolvent.

[0046]FIG. 18 is an isocratic/gradient separation on non-alkylatedpoly(styrene-divinylbenzene) beads.

[0047]FIG. 19 shows a schematic representation of a hybridization toform a mixture of homoduplexes and heteroduplexes.

[0048]FIG. 20 is an elution profile showing separation of a 209 basepair homoduplex/heteroduplex standard mutation detection mixtureperformed by DMIPC at 56° C.

[0049]FIG. 21 is an elution profile of another injection of the same 209bp mixture and using the same column as in FIG. 20, but after changingthe guard cartridge and replacing the pump-valve filter.

[0050]FIG. 22 is an elution profile of another injection of the same 209bp mixture and using the same column as in FIG. 21, but after flushingthe column with 0.1 M TEAA, 25% acetonitrile, and 0.32 M EDTA for 45minutes at 75° C.

[0051]FIG. 23 is a DMIPC elution profile of a 100 bp PCR product from awild-type strand of Lambda DNA.

[0052]FIG. 24 is a DMIPC elution profile of a hybridized mixturecontaining a Lambda DNA strand containing a mutation and wild typestrand.

DETAILED DESCRIPTION OF THE INVENTION

[0053] In its most general form, the subject matter of the presentinvention concerns the separation of polynucleotides. e.g. DNA,utilizing a stationary separation medium having non-polar surfaces. Thepreferred surfaces are essentially free from multivalent cationcontamination which can trap polynucleotides. The separation isperformed on the stationary phase surface. The surface can be porous,but preferably any surface pores are of a size which excludes thesmallest polynucleotide being analyzed.

[0054] The medium can be enclosed in a column. In one embodiment, thenon-polar surfaces comprise the surfaces of beads. In an alternativeembodiment, the surfaces comprise the surfaces of interstitial spaces ina molded monolith. For purposes of simplifying the description of theinvention and not by way of limitation, the separation ofpolynucleotides using nonporous beads, and the preparation of suchbeads, will be primarily described herein, it being understood thatother separation surfaces, such as interstitial surfaces of monoliths,are intended to be included within the scope of this invention.Monoliths such as derivatized silica gel rods contain separation mediawhich have been formed inside a column as a unitary structure havingthrough pores or interstitial spaces which allow eluting solvent andanalyte to pass through and which provide the non-polar separationsurface.

[0055] In general, the only requirement for the separation media of thepresent invention is that they must have a surface that is eitherintrinsically non-polar or be bonded with a material that forms asurface having sufficient non-polarity to interact with a counterionagent.

[0056] In one aspect, the subject matter of the present invention is theseparation of polynucleotides by Matched Ion PolynucleotideChromatography utilizing columns filled with nonporous beads having anaverage diameter of about 0.5-100 microns; preferably, 1-10 microns;more preferably, 1-5 microns. Beads having an average diameter of1.0-3.0 microns are most preferred.

[0057] In U.S. Pat. No. 5,585,236, Bonn, et al., had characterized thepolynucleotide separation process as reverse phase ion pairingchromatography (RPIPC). However, since RPIPC does not incorporatecertain essential characteristics described in the present invention,another term, Matched Ion Polynucleotide Chromatography (MIPC), has beenselected. MIPC as used herein, is defined as a process for separatingsingle and double stranded polynucleotides using non-polar beads,wherein the process uses a counter ion agent, and an organic solvent toelute the polynucleotide from the beads, and wherein the beads arecharacterized as having a DNA Separation Factor of at least 0.05. In apreferred embodiment, the beads are characterized as having a DNASeparation Factor of at least 0.5.

[0058] The performance of the beads of the present invention isdemonstrated by high efficiency separation by MIPC of double strandedand single stranded DNA. Applicants have found that the best criterionfor measuring performance of the beads is a DNA Separation Factor. Thisis measured as the resolution of 257- and 267-base pair double strandedDNA fragments of a pUC18 DNA-HaeIII restriction digest and is defined asthe ratio of the distance from the valley between the peaks to the topof the peaks, over the distance from the baseline to the top of thepeaks. Referring to the schematic representation of FIG. 1, the DNASeparation Factor is determined by measuring the distance “a” from thebaseline to the valley “e” between the peaks “b” and “c” and thedistance “d” from the valley “e” to the top of one of the peaks “b” or“c”. If the peak heights are unequal, the highest peak is used to obtain“d.” The DNA Separation Factor is the ratio of d/(a+d). The peaks of257- and 267-base pairs in this schematic representation are similar inheight. Operational beads of the present invention have a DNA SeparationFactor of at least 0.05. Preferred beads have a DNA Separation Factor ofat least 0.5. In an optimal embodiment, the beads have a DNA SeparationFactor of at least 0.95.

[0059] Without wishing to be bound by theory, Applicants believe thatthe beads which conform to the DNA Separation Factor as specified hereinhave a pore size which essentially excludes the polynucleotides beingseparated from entering the bead. As used herein, the term “nonporous”is defined to denote a bead which has surface pores having a diameterthat is less than the size and shape of the smallest DNA fragment in theseparation in the solvent medium used therein. Included in thisdefinition are beads having these specified maximum size restrictions intheir natural state or which have been treated to reduce their pore sizeto meet the maximum effective pore size required. Preferably, all beadswhich provide a DNA Separation Factor of at least 0.05 are intended tobe included within the definition of “nonporous” beads.

[0060] The surface conformations of nonporous beads of the presentinvention can include depressions and shallow pit-like structures whichdo not interfere with the separation process. A pretreatment of a porousbead to render it nonporous can be effected with any material which willfill the pores in the bead structure and which does not significantlyinterfere with the MIPC process.

[0061] Pores are open structures through which mobile phase and othermaterials can enter the bead structure. Pores are often interconnectedso that fluid entering one pore can exit from another pore. Applicantsbelieve that pores having dimensions that allow movement of thepolynucleotide into the interconnected pore structure and into the beadimpair the resolution of separations or result in separations that havevery long retention times. In MIPC, however, the beads are “nonporous”and the polynucleotides do not enter the bead structure.

[0062] The term polynucleotide is defined as a linear polymer containingan indefinite number of nucleotides, linked from one ribose (ordeoxyribose) to another via phosphoric residues. The present inventioncan be used in the separation of RNA or of double- or single-strandedDNA. For purposes of simplifying the description of the invention, andnot by way of limitation, the separation of double-stranded DNA will bedescribed in the examples herein, it being understood that allpolynucleotides are intended to be included within the scope of thisinvention.

[0063] Chromatographic efficiency of the column beads is predominantlyinfluenced by the properties of surface and near-surface areas. For thisreason, the following descriptions are related specifically to theclose-to-the-surface region of the beads. The main body and/or thecenter of such beads can exhibit entirely different chemistries and setsof physical properties from those observed at or near the surface of thebeads of the present invention.

[0064] In another embodiment of the present invention, the separationmedium can be in the form of a monolith such as a rod-like monolithiccolumn. The monolithic column can be polymerized or formed as a singleunit inside of a tube. The through pore or interstitial spaces providefor the passage of eluting solvent and analyte materials. The separationis performed on the stationary surface. The surface can be porous, butis preferably nonporous. The form and function of the separations areidentical to columns packed with beads. As with beads, the porescontained in the rod must be compatible with DNA and not trap thematerial. Also, the rod must not contain contamination that will trapDNA.

[0065] In one embodiment of the present invention, the separation mediumis continuous monolithic silica gel. A molded monolith can be preparedby polymerization within the confines of a chromatographic column (e.g.,to form a rod) or other containment system. A monolith is preferablyobtained by the hydrolysis and polycondensation of alkoxysilanes. Apreferred monolith is derivatized in order to produce non-polarinterstitial surfaces. Chemical modification of silica monoliths withocatdecyl, methyl or other ligands can be carried out. An example of apreferred derivatized monolith is one which is polyfunctionallyderivatized with octadecylsilyl groups. The preparation of derivatizedsilica monoliths is by conventional methods well known in the art asdescribed in Example 15 and in the following references which are herebyincorporated in their entirety herein: Nakanishi, et al., J. Sol-GelSci. Technol. 8:547 (1997); Nakanishi, et al., Bull, Chem. Soc. Jpn.67:1327 (1994); Cabrera, et al., Trends Analytical Chem. 17:50 (1998);Jinno, et al., Chromatographia 27:288 (1989).

[0066] The beads of the invention comprise a nonporous particle whichhas non-polar molecules or a non-polar polymer attached to or coated onits surface. In general, the beads comprise nonporous particles whichhave been coated with a polymer or which have substantially all surfacesubstrate groups reacted with a non-polar hydrocarbon or substitutedhydrocarbon group, and any remaining surface substrate groups endcappedwith a tri(lower alkyl)chlorosilane or tetra(loweralkyl)dichlorodisilazane as described above.

[0067] The nonporous particle is preferably an inorganic particle, butcan be a nonporous organic particle. The nonporous particle can be, forexample, silica, silica carbide, silica nitrite, titanium oxide,aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides suchas cellulose, or diatomaceous earth, or any of these materials whichhave been modified to be nonporous. Examples of carbon particles includediamond and graphite which have been treated to remove any interferingcontaminants. The preferred particles are essentially non-deformable andcan withstand high pressures. The nonporous particle is prepared byknown procedures. The preferred particle size is about 0.5-100 microns;preferably, 1-10 microns; more preferably, 1-5 microns. Beads having anaverage diameter of 1.0-3.0 microns are most preferred.

[0068] Because the chemistry of preparing conventional silica-basedreverse phase HPLC materials is well-known, most of the description ofthe beads of the invention herein is presented in reference to silica.It is to be understood, however, that other nonporous particles, such asthose listed above, can be modified in the same manner and substitutedfor silica in the process of the invention. For a description of thegeneral chemistry of silica, see Poole, Colin F. and Salwa K. Poole,Chromatography Today, Elsevier:New York (1991), pp. 313-342 and Snyder,R. L. and J. J. Kirkland, Introduction to Modem Liquid Chromatography,2^(nd) ed., John Wiley & Sons, lnc.:New York (1979), pp. 272-278, thedisclosures of which are hereby incorporated herein by reference intheir entireties.

[0069] The nonporous beads of the invention are characterized by havingminimum exposed silanol groups after reaction with the coating orsilating reagents. Minimum silanol groups are needed to reduce theinteraction of the DNA with the substrate and also to improve thestability of the material in a high pH and aqueous environment. Silanolgroups can be harmful because they can repel the negative charge of theDNA molecule, preventing or limiting the interaction of the DNA with thestationary phase of the column. Another possible mechanism ofinteraction is that the silanol can act as ion exchange sites, taking upmetals such as iron (III) or chromium (III). Iron (III) or other metalswhich are trapped on the column can distort the DNA peaks or evenprevent DNA from being eluted from the column.

[0070] Silanol groups can be hydrolyzed by the aqueous-based mobilephase. Hydrolysis will increase the polarity and reactivity of thestationary phase by exposing more silanol sites, or by exposing metalsthat can be present in the silica core. Hydrolysis will be moreprevalent with increased underivatized silanol groups. The effect ofsilanol groups on the DNA separation depends on-which mechanism ofinterference is most prevalent. For example, iron (III) can becomeattached to the exposed silanol sites, depending on whether the iron(III) is present in the eluent, instrument or sample.

[0071] The effect of metals can only occur if metals are already presentwithin the system or reagents. Metals present within the system orreagents can get trapped by ion exchange sites on the silica. However,if no metals are present within the system or reagents, then the silanolgroups themselves can cause interference with DNA separations.Hydrolysis of the exposed silanol sites by the aqueous environment canexpose metals that might be present in the silica core.

[0072] Fully hydrolyzed silica contains a concentration of about 8μmoles of silanol groups per square meter of surface. At best, becauseof steric considerations, a maximum of about 4.5 μmoles of silanolgroups per square meter can be reacted, the remainder of the silanolbeing sterically shielded by the reacted groups. Minimum silanol groupsis defined as reaching the theoretical limit of or having sufficientshield to prevent silanol groups from interfering with the separation.

[0073] Numerous methods exist for forming nonporous silica coreparticles. For example, sodium silicate solution poured into methanolwill produce a suspension of finely divided spherical particles ofsodium silicate. These particles are neutralized by reaction with acid.In this way, globular particles of silica gel are obtained having adiameter of about 1-2 microns. Silica can be precipitated from organicliquids or from a vapor. At high temperature (about 2000° C.), silica isvaporized, and the vapors can be condensed to form finely divided silicaeither by a reduction in temperature or by using an oxidizing gas. Thesynthesis and properties of silica are described by R. K. Iler in TheChemistry of Silica, Solubility, Polymerization, Colloid and SurfaceProperties, and Biochemistry, John Wiley & Sons:New York (1979).

[0074] W. Stöber et al. described controlled growth of monodispersesilica spheres in the micron size range in J. Colloid and InterfaceSci., 26:62-69 (1968). Stöber et al. describe a system of chemicalreactions which permit the controlled growth of spherical silicaparticles of uniform size by means of hydrolysis of alkyl silicates andsubsequent condensation of silicic acid in alcoholic solutions. Ammoniais used as a morphological catalyst. Particle sizes obtained insuspension range from less than 0.05 μm to 2 μm in diameter.

[0075] Nonporous silica core beads can be obtained from Micra Scientific(Northbrook, Ill.) and from Chemie Uetikkon (Lausanne, Switzerland).

[0076] To prepare the nonporous beads of the invention, the nonporousparticle is coated with a polymer or reacted and endcapped so thatsubstantially all surface substrate groups of the nonporous particle areblocked with a non-polar hydrocarbon or substituted hydrocarbon group.This can be accomplished by several methods.

[0077] The organic bonded-phase siloxane coating can be made as amonomolecular layer or as a polymerized multilayer coating. Packingswith so-called monomolecular organic layers are normally prepared byreacting the surface silanol groups of siliceous-base particles withmono-, di-, or trifunctional chloro-, dimethyl-, amino-, siloxy-, oralkoxy-silanes. Typical monofunctional reactants used in these reactionsinclude X—Si—R, where X=Cl, OH, OCH₃, or OC₂H₃, and R is an organicradical. FIG. 2 is a schematic representation of a bead 20 having asilica core 22 and a monomolecular organic layer. (The figure does notnecessarily reflect the morphology or pore structure of the beads of theinvention and is meant for illustrative purposes only.) Using bi- andtrifunctional reactants, such as R₂SiX₂ and RSiX₃, for the surfacemodifications, up to two Si—X groups per bonded functional group remainunreacted. After treatment with water, hydrolysis of these unreactedgroups takes place, and additional silanol groups are formed (sometimesin a polymer matrix) in about the same concentration as the bondedorganic functional groups present in the packing. These acidicorgano-silanol groups can significantly affect the retention behavior ofsolutes and adversely influence the stability of the packing in aqueoussolutions at pH>7.

[0078] Thus, incomplete reaction of the surface with the silane reagent,or the formation of new Si—OH groups from using bi- or trifunctionalmodifiers, can result in a population of residual acidic Si—OH groupsthat are readily accessible to molecules of the mobile phase or sample.Therefore, the recent trend is toward (a) a dense monolayer offunctional groups instead of partial coverage and (b) the use ofmonofunctional dimethylsilanes [X—Si(CH₃)₂-R] to provide a homogeneousorganic coating with a minimum possibility of residual Si—OH groups.Monochlorosilane reagents are preferred, if the required organicfunctionality can be prepared. If two of the R groups in themonofunctional modifier are methyl, surface coverage can be as high asabout 4 μmoles per square meter of organic (based on carbon analysis).In the latter case, residual Si—OH groups on the silica surface areunavailable for chromatographic interactions with most solutes becauseof steric shielding.

[0079] The reaction of organosilanols (e.g., HO—Si—R₃) ororganoalkoxy—(e.g., RO—Si—R₃) silanes with silica supports withoutpolymerization can also produce good packings. These reactions arerelatively reproducible, provided that traces of water or other reactivespecies are absent. Unreacted, accessible silanols can be left after theinitial reaction, but these can be removed by capping of the packingwith chlorotrimethylsilane (providing the R groups do not react with thelatter silane).

[0080] According to one method, the nonporous particle is coated with apolymer coating. Suitable polymers for use in coating the particleinclude chain reaction polymers and step reaction polymers, for example,polystyrene, polymethacrylate, polyethylene, polyurethane,polypropylene, polyamide, insoluble polysaccharides such as cellulose,polydimethyl siloxane, polydialkyl siloxane, and related materials. Thepolymer coating can be attached to the nonporous particle by means of amulti-coating process so that complete shielding of the surface isachieved.

[0081] In the last few years, new bonded phase packings, known aspolymer-coated or polymer-encapsulated packings, have been introducedbased on techniques used to prepare immobilized stationary phases foropen tubular column gas chromatography. In this case, the phases areprepared by mechanically coating either bare silica or presilanizedsilica microparticles with a poly(siloxane) or poly(butadiene)prepolymer, which is then immobilized by peroxide, azo-tert-butane, orgamma radiation-induced chemical crosslinking reactions. FIG. 21 is aschematic illustration of a coated bead 30 having a silica core 32 andpolymer coating 34. (The figure does not necessarily reflect themorphology or pore structure of the beads of the invention and is meantfor illustrative purposes only.)

[0082] An alternative method comprises a combination of covalent bondingwith a vinyl-containing silane molecule and then polymerizing a coatingon the surface of the particles. A second coating can be applied ifresidual silanol groups or metal groups are present.

[0083] In a variation of this method, the silica surface is firstmodified by reaction with vinyltrichlorosilane, followed by polymerizingacrylic acid derivatives to and over the derivatized silica surface. Theavailability of a large number of useful monomers and prepolymers hasenabled a wide variety of reverse phase, polar, and ion exchangepackings to be prepared using the same general reaction. Also, since thegeneral approach does not depend on the chemistry of the underlyingsubstrate, materials other than silica, for example, alumina andzirconia, can be modified and used under conditions for which silica isunsuitable, for example, with mobile phases outside the pH range 2-7.5.Returning to silica, presilanization decreases the number of activesilanol groups, which are then further shielded by the polymeric filmanchored over the surface. In reverse phase liquid chromatography, thesepackings have shown improved chromatographic properties compared tomonomeric, chemically bonded phases for the separation of basic solutes.Polymer-encapsulated packings have a film thickness of about 1 nm tomaintain reasonable mass transfer characteristics. A description of thisprocedure has been published by H. Engelhart et al. (Chromatographia,27:535 (1989)).

[0084] The polymer-coated beads prepared according to either of theabove methods can be used in their unmodified state or can be modifiedby substitution with a hydrocarbon group. Any hydrocarbon group issuitable. The term “hydrocarbon” as used herein is defined to includealkyl and alkyl substituted aryl groups, having from 1 to 1,000,000carbons, the alkyl groups including straight chained, branch chained,cyclic, saturated, unsaturated nonionic functional groups of varioustypes including, aldehyde, ketone, ester, ether, alkyl groups, and thelike, and the aryl groups including as monocyclic, bicyclic, andtricyclic aromatic hydrocarbon groups including phenyl, naphthyl, andthe like. Methods for hydrocarbon substitution are conventional andwell-known in the art and are not an aspect of this invention. Thehydrocarbon can also contain hydroxy, cyano, nitro groups, or the likewhich are considered to be non-polar, reverse phase functional groups.The preferred hydrocarbon groups are alkyl groups, and the descriptionof suitable substitution processes hereinbelow are presented asalkylation for purposes of simplification and not by way of limitation,it being understood that aryl substitution by conventional proceduresare also intended to be included within the scope of this invention.

[0085] The polymer-coated beads can be alkylated by reaction with thecorresponding alkyl halide such as the alkyl iodide. Alkylation isachieved by mixing the polymer-coated beads with an alkyl halide in thepresence of a Friedel-Crafts catalyst to effect electrophilic aromaticsubstitution on the aromatic rings at the surface of the polymer blend.Suitable Friedel-Crafts catalysts are well-known in the art and includeLewis acids such as aluminum chloride, boron trifluoride, tintetrachloride, etc. Substitution with hydrocarbon groups having from 1to 1,000,000 and preferably from 1 to 22 carbons can be effected bythese processes. Hydrocarbon groups having from 23 to 1,000,000 carbonsare referenced herein as hydrocarbon polymers.

[0086] Alkylation can be accomplished by a number of known synthesisprocedures. These include Friedel-Crafts alkylation with an alkylhalide, attachment of an alkyl alcohol to a chloromethylated bead toform an ether, etc. Although the preferred method for alkylating thepolymer-coated beads of the present invention is alkylation after thepolymer coating has been formed on the nonporous particle, analternative method of alkylation is to polymerize alkylated monomers toform an alkylated polymer coating on the nonporous particle. In thisembodiment, the monomers will be substituted with alkyl groups havingany number of carbon atoms, for example, from 1 to 100, 1 to 50 or 1 to24, for example, depending upon the requirements of the separationvariables.

[0087] As an alternative to polymer coating, the nonporous particle canbe functionalized with an alkyl group or other non-polar functionalgroup including cyano, ester, and other non-ionic groups, followed by acomplete endcapping process to reduce silanol and metal interaction.Endcapping of the nonporous particle can be achieved by reacting theparticle with trialkyl chlorosilane or tetraalkyl dichlorodisilazane,such as, for example, trimethyl chlorosilane ordichloro-tetraisopropyl-disilazane.

[0088] A large number of factors influence the success of the bondingreactions and the quality of the final bonded-phase product. The rateand extent of the bonding reaction depends on the reactivity of thesilane, choice of solvent and catalyst, time, temperature, and the ratioof reagents to substrate. Reactive organosilanes with Cl, OH, OR,N(CH₃)₂, OCOCF₃, and enolates as leaving groups have been widely used.The dimethylamine, trifluoroacetate, and enol ethers ofpentane-2,4-dione are the most reactive leaving groups, althougheconomy, availability, and familiarity result in the chlorosilanes andalkoxysilanes being the most widely used, particularly among commercialmanufacturers. Initially, reactions can be almost stoichiometric but, asthe surface coverage approaches a maximum value, the reaction becomesvery slow. For this reason, reaction times tend to be long (12-72hours), reaction temperatures moderately high (in most cases, around100° C.) and, in the case of chlorosilanes, an acid acceptor catalyst(e.g., pyridine) is used. Some reagents, such as the alkylsilyl enolatesand alkylsilyldimethylamines, do not require additional catalyst, oreven solvent, to carry out the reaction. The most common solventsemployed are toluene and xylene, although other solvents, such as carbontetrachloride, trichloroethane, and dimethylformamide (DMF), have beenrecommended as being superior. Since the bonding reactions are carriedout by refluxing in an inert atmosphere, solvents are often selectedbased on their capacity to be a good solvent for the organosilanes andto attain the desired reaction temperature at reflux. Except for3-cyanopropylsiloxane bonded phases, the high reactivity ofchlorosilanes towards certain polar functional groups (e.g., OH, etc.)precludes the use of these groups for the preparation of polar, reversephase bonded phases. Alkoxysilanes containing acidic or basic functionalgroups are autocatalytic and the bonded phases are usually prepared byrefluxing the silane in an inert solvent at a temperature high enough todistill off the alcohol formed by the condensation reaction with thesurface silanol groups. Bonding of neutral, polar ligands generallyrequires the addition of a catalyst, such as toluene-4-sulfonic acid ortriethylamine, in the presence of sufficient water to generate monolayercoverage of the silica. The presence of water speeds up the hydrolysisof the alkoxy groups of the adsorbed organosilane, which tends to reactwith surface silanol groups rather than polymerize in solution. It seemsto be a general problem in the preparation of polar bonded phases thatsurface silanol groups are blocked by physically adsorbed organosilanes,giving rise to a lower bonded phase density after workup than themaximum theoretically predicted. The bonded phase density can beincreased by repeating the reaction a second time or exposed silanolgroups minimized by endcapping.

[0089] Although most bonded phases are prepared from organosilanescontaining a single functionalized ligand bonded to silicon, with theremaining groups being leaving groups and/or methyl groups, more highlysubstituted organosilanes can also be used. Bifunctional organosilanes,such as 1,3-dichlorotetraisopropyldisilazane, are able to react withsurface silanol groups at both ends of the chain, forming a bonded phasethat is more hydrolytically stable than bonded phases formed fromconventional organosilanes. The bidentate organosilanes have reactivesites that more closely match the spacing of the silanol groups on thesilica surface and provide a higher bonded phase coverage than isachieved with dichlorosilanes with both leaving groups attached to thesame silicon atom. For alkyldimethylsilanes, increasing the length ofthe alkyl group increases the hydrolytic stability of the bonded phaserelative to that of the trimethylsilyl bonded ligands. Increasing thechain length of the methyl groups increases the hydrolytic stability ofthe bonded phase, but reduces the phase coverage due to steric effects.The use of monofunctional organosilanes containing one or two bulkygroups, for example, isopropyl or t-butyl, on the silicon atom of thesilane can become more important in the preparation of bonded phases foruse at low pH. The bulky alkyl groups provide better steric protectionto the hydrolytically sensitive siloxane groups on the packing surfacethan does the methyl group.

[0090] The general process of coating and endcapping of a silicasubstrate is well-known technology. However, the general understandingof those who have used these materials is they are not suitable for highperformance double stranded DNA separations. However, the beads of thisinvention are formed by a more careful application of the coating andend-capping procedures to effect a thorough shielding of the silicacore, the resulting beads having the ability to perform rapidseparations of both single stranded and double stranded DNA which areequal to or better than those achieved using the alkylated nonporouspolymer beads disclosed in U.S. Pat. No. 5,585,236, for example.

[0091] Care must be taken during the preparation of the beads to ensurethat the surface of the beads has minimum silanol or metal oxideexposure and that the surface remains nonporous.

[0092] In an important aspect of the present invention, the beads orother media of the invention are characterized by having low amounts ofmetal contaminants or other contaminants that can bind DNA. Thepreferred beads are characterized by having been subjected toprecautions during production, including a decontamination treatment,such as an acid wash treatment, designed to substantially eliminate anymultivalent cation contaminants (e.g. Fe(III), Cr(III), or colloidalmetal contaminants). Only very pure, non-metal containing materialsshould be used in the production of the beads in order that theresulting beads will have minimum metal content.

[0093] In addition to the beads themselves being substantiallymetal-free, Applicants have also found that, to achieve optimum peakseparation during RPIPC, the separation column and all process solutionsheld within the column or flowing through the column are preferablysubstantially free of multivalent cation contaminants (e.g. Fe(III),Cr(III), and colloidal metal contaminants). As described in commonlyowned U.S. Pat. No. 5,772,889 to Gjerde (1998), and in commonly owned,co-pending U.S. patent applications Ser. No. 09/081,040 filed May 18,1998 and Ser No. 09/080,547 filed May 18, 1998, all three of which areincorporated by reference herein, this can be achieved by supplying andfeeding solutions that enter the separation column with components whichhave process solution-contacting surfaces made of material which doesnot release multivalent cations into the process solutions held withinor flowing through the column, in order to protect the column frommultivalent cation contamination. The process solution-contactingsurfaces of the system components are preferably material selected fromthe group consisting of titanium, coated stainless steel, passivatedstainless steel, and organic polymer. Metals found in stainless steel,for example, do not harm the separation, unless they are in an oxidizedor colloidal partially oxidized state. For example, 316 stainless steelfrits are acceptable in column hardware, but surface oxidized stainlesssteel frits harm the DNA separation.

[0094] For additional protection, multivalent cations in mobile phasesolutions and sample solutions entering the column can be removed bycontacting these solutions with multivalent cation capture resin beforethe solutions enter the column to protect the separation medium frommultivalent cation contamination. The multivalent capture resin ispreferably cation exchange resin and/or chelating resin.

[0095] Mixtures of polynucleotides in general, and double stranded DNAin particular, are effectively separated using Matched IonPolynucleotide Chromatography (MIPC). MIPC separations ofpolynucleotides at non-denaturing temperature, typically less than about50° C., are based on base pair length. However, even traces ofmultivalent cations anywhere in the solvent flow path can cause asignificant deterioration in the resolution of the separation aftermultiple uses of an MIPC column. This can result in increased costcaused by the need to purchase replacement columns and increaseddowntime.

[0096] Therefore, effective measures are preferably taken to preventmultivalent metal cation contamination of the separation systemcomponents, including separation media and mobile phase contacting.These measures include, but are not limited to, washing protocols toremove traces of multivalent cations from the separation media andinstallation of guard cartridges containing cation capture resins, inline between the mobile phase reservoir and the MIPC column. These, andsimilar measures, taken to prevent system contamination with multivalentcations have resulted in extended column life and reduced analysisdowntime.

[0097] There are two places where multivalent cation binding agents,e.g., chelators, are used in MIPC separations. In one embodiment, thesebinding agents can be incorporated into a solid through which the mobilephase passes. Contaminants are trapped before they reach places withinthe system that can harm the separation. In these cases, the functionalgroup is attached to a solid matrix or resin (e.g., a flow-throughcartridge, usually an organic polymer, but sometimes silica or othermaterial). The capacity of the matrix is preferably about 2 meq/g. Anexample of a suitable chelating resin is available under the trademarkCHELEX 100 (Dow Chemical Co.) containing an iminodiacetate functionalgroup.

[0098] In another embodiment, the multivalent cation binding agent canbe added to the mobile phase. The binding functional group isincorporated into an organic chemical structure. The preferredmultivalent cation binding agent fulfills three requirements. First, itis soluble in the mobile phase. Second, the complex with the metal issoluble in the mobile phase. Multivalent cation binding agents such asEDTA fulfill this requirement because both the chelator and themultivalent cation binding agent-metal complex contain charges whichmake them both water-soluble. Also, neither precipitate whenacetonitrile, for example, is added. The solubility in aqueous mobilephase can be enhanced by attaching covalently bound ionic functionality,such as, sulfate, carboxylate, or hydroxy. A preferred multivalentcation binding agent can be easily removed from the column by washingwith water, organic solvent or mobile phase. Third, the binding agentmust not interfere with the chromatographic process.

[0099] The multivalent cation binding agent can be a coordinationcompound. Examples of preferred coordination compounds include watersoluble chelating agents and crown ethers. Non-limiting examples ofmultivalent cation binding agents which can be used in the presentinvention include acetylacetone, alizarin, aluminon, chloranilic acid,kojic acid, morin, rhodizonic acid, thionalide, thiourea,α-furildioxime, nioxime, salicylaldoxime, dimethylglyoxime,α-furildioxime, cupferron, (α-nitroso-β-naphthol, nitroso-R-salt,diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN,SPADNS, glyoxal-bis(2-hydroxyanil), murexide, α-benzoinoxime, mandelicacid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine,thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids,α,α′-bipyridine, 4-hydroxybenzothiazole, 8-hydroxyquinaldine,8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid,(α,α′,α″-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,salicylic acid, tiron, 4-chloro-1,2-dimercaptobenzene, dithiol,mercaptobenzothiazole, rubeanic acid, oxalic acid, sodiumdiethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate. These andother examples are described by Perrin in Organic Complexing Reagents:Structure, Behavior, and Application to Inorganic Analysis, Robert E.Krieger Publishing Co. (1964). In the present invention, a preferredmultivalent cation binding agent is EDTA.

[0100] To achieve high resolution chromatographic separations ofpolynucleotides, it is generally necessary to tightly pack thechromatographic column with the solid phase nonporous beads. Any knownmethod of packing the column with a column packing material can be usedin the present invention to obtain adequate high resolution separations.Typically, a slurry of the beads is prepared using a solvent having adensity equal to or less than the density of the beads. The column isthen filled with the bead slurry and vibrated or agitated to improve thepacking density of the beads in the column. Mechanical vibration orsonification are typically used to improve packing density.

[0101] For example, to pack a 50×4.6 mm i.d. column, 2.0 grams of beadscan be suspended in 10 mL of methanol with the aid of sonification. Thesuspension is then packed into the column using 50 mL of methanol at8,000 psi of pressure. This improves the density of the packed bed.

[0102] The separation method of the invention is generally applicable tothe chromatographic separation of single stranded and double strandedpolynucleotides of DNA and RNA. Samples containing mixtures ofpolynucleotides can result from total synthesis of polynucleotides,cleavage of DNA or RNA with restriction endonucleases or with otherenzymes or chemicals, as well as polynucleotide samples which have beenmultiplied and amplified using polymerase chain reaction techniques.

[0103] The method of the present invention can be used to separatedouble stranded polynucleotides having up to about 1500 to 2000 basepairs. In many cases, the method is used to separate polynucleotideshaving up to 600 bases or base pairs, or which have up to 5 to 80 basesor base pairs.

[0104] In a preferred embodiment, the separation is by MIPC. Thenonporous beads of the invention are used as a reverse phase materialthat will function with counter ion agents and a solvent gradient toeffect the DNA separations. In MIPC, the DNA fragments are matched witha counterion agent and then subjected to reverse phase chromatographyusing the nonporous beads of the present invention.

[0105] There are several types of counterions suitable for use withMIPC. These include a mono-, di-, or trialkylamine that can beprotonated to form a positive counter charge or a quaternary alkylsubstituted amine that already contains a positive counter charge. Thealkyl substitutions may be uniform (for example, triethylammoniumacetate or tetrapropylammonium acetate) or mixed (for example,propyldiethylammonium acetate). The size of the alkyl group may be small(methyl) or large (up to 30 carbons) especially if only one of thesubstituted alkyl groups is large and the others are small. For exampleoctyldimethylammonium acetate is a suitable counterion agent. Preferredcounterion agents are those containing alkyl groups from the ethyl,propyl or butyl size range.

[0106] The purpose of the alkyl group is to impart a nonpolar characterto the polynucleic acid through a matched ion process so that thepolynucleic acid can interact with the nonpolar surface of theseparation media. The requirements for the extent of nonpolarity of thecounterion-DNA pair depends on the polarity of the separation media, thesolvent conditions required for separation, and the particular size andtype of fragment being separated. For example, if the polarity of theseparation media is increased, then the polarity of the counterion agentmay have to change to match the polarity of the surface and increaseinteraction of the counterion-DNA pair. Triethylammonium acetate ispreferred although quaternary ammonium reagents such as tetrapropyl ortetrabutyl ammonium salts can be used when extra nonpolar character isneeded or desired. In general, as the polarity of the alkyl group isincreased, size specific separations, sequence independent separationsbecome more possible. Quaternary counterion reagents are not volatile,making collection of fragments more difficult.

[0107] The mobile phase preferably contains a counterion agent. Typicalcounterion agents include trialkylammonium salts of organic or inorganicacids, such as lower alkyl primary, secondary, and lower tertiaryamines, lower trialkyammonium salts and lower quaternary alkyalmmoniumsalts. Lower alkyl refers to an alkyl radical of one to six carbonatoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl,isoamyl, n-pentyl, and isopentyl. Examples of counterion agents includeoctylammonium acetate, octadimethylammonium acetate, decylammoniumacetate, octadecylammonium acetate, pyridiniumammonium acetate,cyclohexylammonium acetate, diethylammonium acetate, propylethylammoniumacetate, propyldiethylammonium acetate, butylethylammonium acetate,methylhexylammonium acetate, tetramethylammonium acetate,tetraethylammonium acetate, tetrapropylammonium acetate,tetrabutylammonium acetate, dimethydiethylammonium acetate,triethylammonium acetate, tripropylammonium acetate, andtributylammonium acetate. Although the anion in the above examples isacetate, other anions may also be used, including carbonate, phosphate,sulfate, nitrate, propionate, formate, chloride, and bromide, or anycombination of cation and anion. These and other agents are described byGjerde, et al. in Ion Chromatography, 2nd Ed., Dr. Alfred Hüthig VerlagHeidelberg (1987). Counterion agents that are volatile are preferred foruse in the method of the invention, with triethylammonium acetate (TEAA)and triethylammonium hexafluoroisopropyl alcohol being most preferred.

[0108] To achieve optimum peak resolution during the separation of DNAby MIPC using the beads of the invention, the method is performed at atemperature within the range of 20° C. to 90° C. to yield aback-pressure not greater than 10,000 psi. In general, separation ofsingle-stranded fragments should be performed at higher temperatures.

[0109] Applicants have found that the temperature at which theseparation is performed affects the choice of organic solvents used inthe separation. When the separation is performed at a temperature withinthe above range, an organic solvent that is water soluble is preferablyused, for example, alcohols, nitrites, dimethylformamide (DMF), esters,and ethers. Water soluble solvents are defined as those which exist as asingle phase with aqueous systems under all conditions of operation ofthe present invention. Solvents which are particularly preferred for usein the method of this invention include methanol, ethanol, 1-propanol,2-propanol, tetrahydrofuran (THF), and acetonitrile, with acetonitrilebeing most preferred. In some cases, it may be desired to increase therange of concentration of organic solvent used to perform theseparation. For example, increasing the alkyl length on the counterionagent will increase the nonpolarity of the counterion-DNA pair resultingin the need to either increase the concentration of the mobile phaseorganic solvent, or increase the strength of the organic solvent type,e.g. acetonitrile is about two times more effective than methanol foreluting polynucleic acids. There is a positive correlation betweenconcentration of the organic solvent required to elute a fragment fromthe column and the length of the fragment. However, at high organicsolvent concentrations, the polynucleotide could precipitate. To avoidprecipitation, a strong organic solvent or a smaller counterion alkylgroup can be used. The alkyl group on the counterion reagent can also besubstituted with halides, nitro groups, or the like to moderatepolarity.

[0110] Applicants have determined that the chromatographic separationsof double stranded DNA fragments exhibit unique Sorption Enthalpies(ΔH_(sorp)). Two compounds (in this case, DNA fragments of differentsize) can only be separated if they have different partitioncoefficients (K). The Nernst partition coefficient is defined as theconcentration of an analyte (A) in the stationary phase divided by itsconcentration in the mobile phase:$K = \frac{\lbrack A\rbrack_{s}}{\lbrack A\rbrack_{m}}$

[0111] The partition coefficient (K) and the retention factor (k) arerelated through the following equations:$K = {{\frac{{n(A)}_{s}V_{m}}{{n(A)}_{m}V_{s}}\quad {and}\quad k} = \frac{{n(A)}_{s}}{{n(A)}_{m}}}$

[0112] the quotient V_(m)/V_(s) is also called phase volume ratio (Φ).Therefore:

k=KΦ

[0113] To calculate the sorption enthalpies, the following fundamentalthermodynamic equations are necessary:${{\ln \quad K} = {- \frac{\Delta \quad G}{RT}}},{{\ln \quad k} = {{{- \frac{\Delta \quad G_{sorp}}{RT}} + {\ln \quad \Phi \quad {and}\quad \Delta \quad G_{sorp}}} = {{\Delta \quad H_{sorp}} - {T\quad \Delta \quad S_{sorp}}}}}$

[0114] By transforming the last two equations, one obtains the Van'tHoff equation:${\ln \quad k} = {{- \frac{\Delta \quad H_{sorp}}{RT}} + \frac{\Delta \quad S_{sorp}}{R} + {\ln \quad \Phi}}$

[0115] From a plot In k versus 1/T, the sorption enthalpy ΔH_(sorp) canbe obtained from the slope of the graph (if a straight line isobtained). ΔS_(sorp) can be calculated if the phase volume ratio (Φ) isknown.

[0116] Experiments on polymeric beads coated withpoly(styrene-divinylbenzene) also give a negative slope for a plot of Ink versus 1/T, although the plot is slightly curved.

[0117] If the acetonitrile is replaced with methanol, the retentionfactor k decreases with increasing temperature, indicating the retentionmechanism is an exothermic process (ΔH_(sorp)<0).

[0118] The thermodynamic data (as shown in the Examples hereinbelow)reflect the relative affinity of the DNA-counter ion agent complex forthe beads of the invention and the elution solvent. An endothermic plotindicates a preference of the DNA complex for the bead. An exothermicplot indicates a preference of the DNA complex for the solvent over thebead. The plots shown herein are for alkylated and non-alkylatedsurfaces as described in the Examples. Most liquid chromatographicseparations show exothermic plots.

[0119] Recently, MIPC has been successfully applied to the detection ofmutations in double stranded DNA by separating heteroduplexes fromhomoduplexes as described in co-pending U.S. patent application No.09/129,105 filed Aug. 4, 1998 which is herein incorporated by reference.Such separations depend on the lower temperature required to denature aheteroduplex at the site of base pair mismatch compared to a fullycomplimentary homoduplex DNA fragment. MI PC, when performed at atemperature which is sufficient to partially denature a heteroduplex isreferred to herein as Denaturing Matched Ion PolynucleotideChromatography (DMIPC). DMIPC is typically performed at a temperaturebetween 52° C. and 70° C. The optimum temperature for performing DMIPCis 54° C. to 59° C.

[0120] The precautions described hereinabove taken to remove multivalentmetal cations were adequate for maintaining column life, as demonstratedby good separation efficiency, under non-denaturing conditions. However,Applicants have surprisingly found that when performed at partiallydenaturing temperature, conditions for effective DMIPC separationsbecome more stringent. For example, a separation of a standard pUC18HaeIII digest on a MIPC column at 50° C. provided a good separation ofall the DNA fragments in the digest. However, a standard 209 bp DYS271mutation detection mixture of homoduplexes and heteroduplexes, preparedas described in Example 15, applied to the same MIPC column and elutedunder DMIPC conditions, i.e., 56° C., afforded a poor separation of themixture components. In order to optimize column life and maintaineffective separation performance of homoduplexes from heteroduplexes atpartially denaturing temperatures, as is required for mutationdetection, special column washing and storage procedures are used in theembodiments of the invention as described hereinbelow.

[0121] In one aspect of this invention, therefore, an aqueous solutionof multivalent cation binding agent is flowed through the column tomaintain separation efficiency. In order to maintain the separationefficiency of a MIPC column, the column is preferably washed withmultivalent cation binding agent solution after about 500 uses or whenthe performance starts to degrade. Examples of suitable cation bindingagents are as described hereinabove.

[0122] The concentration of a solution of the cation binding agent canbe between 0.01M and 1M. In a preferred embodiment, the column washingsolution contains EDTA at a concentration of about 0.03 to 0.1M.

[0123] In another embodiment, the solution contains an organic solventselected from the group consisting of acetonitrile, ethanol, methanol,2-propanol, and ethyl acetate. A preferred solution contains at least 2%organic solvent to prevent microbial growth. In a most preferredembodiment a solution containing 25% acetonitrile is used to wash a MIPCcolumn. The multivalent cation binding solution can contain a counterionagent as described hereinabove.

[0124] In one embodiment of a column washing procedure, the MIPCseparation column is washed with the multivalent cation binding solutionat an elevated temperature in the range of 50° C. to 80° C. In apreferred embodiment the column is washed with a solution containingEDTA, TEAA, and acetonitrile, in the 70° C. to 80° C. temperature range.In a specific embodiment, the solution contains 0.032 M EDTA, 0.1M TEAA,and 25% acetonitrile.

[0125] Column washing can range from 30 seconds to one hour. Forexample, in a high throughput DMIPC assay, the column can be washed for30 seconds after each sample, followed by equilibration with mobilephase. Since DMIPC can be automated by computer, the column washingprocedure can be incorporated into the mobile phase selection programwithout additional operator involvement. In a preferred procedure, thecolumn is washed with multivalent cation binding agent for 30 to 60minutes at a flow rate preferably in the range of about 0.05 to 1.0mL/min.

[0126] In one embodiment, a MIPC column is tested with a standardmutation detection mixture of homoduplexes and heteroduplexes afterabout 1000 sample analyses. If the separation of the standard mixturehas deteriorated compared to a freshly washed column, then the columncan be washed for 30 to 60 minutes with the multivalent cation bindingsolution at a temperature above about 50° C. to restore separationperformance.

[0127] Applicants have found that other treatments for washing a columncan also be used alone or in combination with those indicatedhereinabove. These include: use of high pH washing solutions (e.g., pH10-12), use of denaturants such as urea or formamide, and reverseflushing the column with washing solution.

[0128] In another aspect, Applicants have discovered that columnseparation efficiency can be preserved by storing the column separationmedia in the column containing a solution of multivalent cation bindingagent therein. The solution of binding agent may also contain acounterion agent. Any of the multivalent cation binding agents,counterion agents, and solvents described hereinabove are suitable forthe purpose of storing a MIPC column. In a preferred embodiment, acolumn packed with MIPC separation media is stored in an organic solventcontaining a multivalent cation binding agent and a counterion agent. Anexample of this preferred embodiment is 0.032 M EDTA and 0.1M TEAA in25% aqueous acetonitrile. In preparation for storage, a solution ofmultivalent cation binding agent, as described above, is passed throughthe column for about 30 minutes. The column is then disconnected fromthe HPLC apparatus and the column ends are capped with commerciallyavailable threaded end caps made of material which does not releasemultivalent cations. Such end caps can be made of coated stainlesssteel, titanium, organic polymer or any combination thereof.

[0129] The effectiveness of the surprising discovery made by Applicants,that washing a MIPC column with a multivalent cation binding agentrestores the ability of the column to separate heteroduplexes andhomoduplexes in mutation detection protocols under DMIPC conditions, isdescribed in Example 18 and demonstrated in FIGS. 20, 21, and 22. Asdescribed in Example 18, Applicants noticed a decrease in resolution ofhomoduplexes and heteroduplexes during the use of a MIPC column inmutation detection. However, no apparent degradation in resolution wasobserved when a DNA standard containing pUC18 HaeIII digest(Sigma/Aldrich Chemical Co.) was applied at 50° C. (not shown). In orderto further test the column performance, a mixture of homoduplexes andheteroduplexes in a 209 bp DNA standard was applied to the column underDMIPC conditions of 56° C. (Kuklin et al., Genetic Testing 1:201 (1998).It was surprisingly observed the peaks representing the homoduplexes andheteroduplexes of the mutation detection standard were poorly resolved(FIG. 20).

[0130]FIG. 21 shows some improvement in the separation of homoduplexesand heteroduplexes of the standard mutation detection mixture when aguard cartridge containing cation capture resin was deployed in linebetween the solvent reservoir and the MIPC system. The chromatographyshown in FIG. 21 was performed at 56° C. The column used in FIG. 21 wasthe same column used in the separation shown in FIG. 20 and forseparating the standard pUC18 HaeIII digest.

[0131]FIG. 22 shows the separation of homoduplexes and heteroduplexes ofthe standard mutation detection mixture at 56° C. on the same columnused to generate the chromatograms in FIGS. 20 and 21. However, in FIG.22 the column was washed for 45 minutes with a solution comprising 32 mMEDTA and 0.1M TEAA in 25% acetonitrile at 75° C. prior to sampleapplication. FIG. 22 shows four cleanly resolved peaks representing thetwo homoduplexes and the two heteroduplexes of the standard 209 bpmutation detection mixture. This restoration of the separation ability,after washing with a solution containing a cation binding agent, of theMIPC column under DMIPC conditions compared to the chromatograms ofFIGS. 20 and 21 clearly shows the effectiveness and the utility of thepresent invention.

[0132] In an important aspect of the present invention, Applicants havedeveloped a standardized criteria to evaluate the performance of a DMIPCseparation media. DMIPC as used herein, is defined as a process forseparating heteroduplexes and homoduplexes using a non-polar separationmedium (e.g., beads or rod) in the column, wherein the process uses acounterion agent, and an organic solvent to desorb the nucleic acid fromthe medium, and wherein the medium is characterized as having a MutationSeparation Factor (MSF) of at least 0.1. In one embodiment, the mediumhas a Mutation Separation Factor of at least 0.2. In a preferredembodiment, the medium has a Mutation Separation Factor of at least 0.5.In an optimal embodiment, the medium has a Mutation Separation Factor ofat least 1.0.

[0133] The performance of the column is demonstrated by high efficiencyseparation by DMIPC of heteroduplexes and homoduplexes. Applicants havefound that the best criterion for measuring performance is a MutationSeparation Factor as described in Example 17. This is measured as thedifference between the areas of the resolved heteroduplex and homoduplexpeaks. A correction factor may be applied to the generated areasunderneath the peaks. The following aspects may affect the calculatedareas of the peaks and reproducibility of the same: baseline drawn, peaknormalization, inconsistent temperature control, inconsistent elutionconditions, detector instability, flow rate instability, inconsistentPCR conditions, and standard and sample degradation. Some of theseaspects are discussed by Snyder, et al., in Introduction to ModernLiquid Chromatography, 2^(nd) Ed., John Wiley and Sons, pp. 542-574(1979) which is incorporated by reference herein.

[0134] The Mutation Separation Factor (MSF) is determined by thefollowing equation:

MSF=(area peak 2−area peak 1)/area peak 1

[0135] where area peak 1 is the area of the peak measured after DMIPCanalysis of wild type and area peak 2 is the total area of the peak orpeaks measured after DMIPC analysis of a hybridized mixture containing aputative mutation, with the hereinabove correction factors taken intoconsideration, and where the peak heights have been normalized to thewild type peak height. Separation particles are packed in an HPLC columnand tested for their ability to separate a standard hybridized mixturecontaining a wild type 100 bp Lambda DNA fragment and the corresponding100 bp fragment containing an A to C mutation at position 51.

[0136] High pressure pumps are used for pumping mobile phase in thesystems described in U.S. Pat. No. 5,585,236 to Bonn and in U.S. Pat.No. 5,772,889 to Gjerde. It will be appreciated that other methods areknown for driving mobile phase through separation media and can be usedin carrying out the separations of polynucleotides as described in thepresent invention. A non-limiting example of such an alternative methodincludes “capillary electrochromatography” (CEC) in which an electricfield is applied across capillary columns packed-with microparticles andthe resulting electroosmotic flow acts as a pump for chromatography.Electroosmosis is the flow of liquid, in contact with a solid surface,under the influence of a tangentially applied electric field. Thetechnique combines the advantages of the high efficiency obtained withcapillary electrophoretic separations, such as capillary zoneelectrophoresis, and the general applicability of HPLC. CEC has thecapability to drive the mobile phase through columns packed withchromatographic particles, especially small particles, when usingelectroosmotic flow. High efficiencies can be obtained as a result ofthe plug-like flow profile. In the use of CEC in the present invention,solvent gradients are used and rapid separations can be obtained usinghigh electric fields. The following references describing CEC are eachincorporated in their entirety herein: Dadoo, et al, LC-GC 15:630(1997); Jorgenson, et al., J. Chromatog. 218:209 (1981); Pretorius, etal., J. Chromatog. 99:23 (1974); and the following U.S. Pat. Nos. toDadoo 5,378,334 (1995), 5,342,492 (1994), and 5,310,463 (1994). In theoperation of this aspect of the present invention, the capillaries arepacked, either electrokinetically or using a pump, with the separationbeads described in the present specification. In another embodiment, apolymeric rod is prepared by bulk free radical polymerization within theconfines of a capillary column. Capillaries are preferably formed fromfused silica tubing or etched into a block. The packed capillary (e.g.,a 150-μm i.d. with a 20-cm packed length and a window locatedimmediately before the outlet frit) is fitted with frits at the inletand outlet ends. An electric field, e.g., 2800V/cm, is applied.Detection can be by uv absorbance or by fluorescence. A gradient oforganic solvent, e.g., acetonitrile, is applied in a mobile phasecontaining counterion agent (e.g. 0.1 M TEAA). to elute thepolynucleotides. The column temperature is maintained by conventionaltemperature control means. In the preferred embodiment, all of theprecautions for minimizing trace metal contaminants as describedhereinabove are employed in using CEC.

[0137] In a related method, mixtures of polynucleotides are separated onthin layer chromatography (TLC) plates. In this method, the beads of thepresent invention are mixed with a binder and bound to a TLC plate byconventional methods (Remington: The Science and Practice of Pharmacy,19^(th) Edition, Gennaro ed., Mack Publishing Co. (1995) pp. 552-554). Afluorophore is optionally included in the mixture to facilitatedetection. The sample is spotted on the plate and the sample is runisocratically under capillary flow. In a preferred embodiment, thesample is run under electroosmotic flow in a process called High-SpeedTLC (HSTLC). In the case of HSTLC, the plate is first wetted withsolvent (e.g., acetonitrile solution in the presence of counterionagent) and an electric field (e.g., 2000 V/cm) is applied. Solventaccumulating at the top of the plate is removed by suction.

[0138] Applicants have surprisingly discovered that dsDNA of selectedranges of base pair length are separable under isocratic conditions byMIPC using the beads of the present invention as described in Example 6.The isocratic mobile phase conditions for separating a selected range ofDNA base pair length, as determined using MIPC, are used in the TLC andHSTLC methods.

[0139] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

[0140] All references cited herein are hereby incorporated by referencein their entirety.

[0141] Procedures described in the past tense in the examples below havebeen carried out in the laboratory. Procedures described in the presenttense have not been carried out in the laboratory, and areconstructively reduced to practice with the filing of this application.

EXAMPLE 1 C-18 Bonded Phase Standard Phase

[0142] To a 1000-mL round bottomed flask, add 200 g of nonporous, 2 μmsilica and one small stirring egg. Transfer flask with silica to an ovenand heat at 125° C. overnight (i.e., at least 8 hours). Have heatingmantle and condenser set up.

[0143] The C-18 bonding reagent, n-octadecyldimethylsilane, is a waxywhite solid to semi-solid at room temperature. To transfer, open thebottle in a hood and gently warm with a heat gun (note: pressure canbuild up in stored chlorosilane bottles, and they should be handled asif they were HCI, as upon contact with moisture, HCI is the sideproduct).

[0144] To a second flask, transfer 125 g of then-octadecylmethylchlorosilane reagent, 10 mL of chloroform, 400 mL oftoluene, and 65 mL of pyridine. Mix the liquid reagents by swirling, andthen add to the dried silica and swirl until all of the silica issuspended. Attach the reflux condenser and bring the mixture to refluxfor 15 hours. Let the mixture cool, such that refluxing has stopped. Addthe capping reagent package of 20 mL of trimethylchlorosilane, 6 mL ofhexamethylsilane in 20 mL of toluene. Resuspend the mixture and bringthe system back to reflux for 6 hours. Let the mixture cool to roomtemperature.

[0145] Transfer to a Buchner funnel and wash with three 200-mL aliquotsof methanol, followed by three 200-mL aliquots of acetone. Air dry forat least 0.5 hour, and then dry in the oven at 100° C. overnight.

[0146] Submit sample for elemental analysis, and percent carbon.

[0147] Dried bonded phase is now ready for column packing.

EXAMPLE 2 CN Bonded Phase, Cyano Phase

[0148] To a 1000-mL round-bottomed flask, add 200 g of nonporous, 2 μmsilica, one stirring egg, and place in an oven at 125° C. overnight(i.e., at least 8 hours) to dry. To the dried silica, add 100 mL of the3-cyanopropylmethyldichlorosilane, 10 mL of chloroform, 450 mL oftoluene, and 50 mL of pyridine. Suspend the mixture and bring to refluxfor 15 hours. Cool filter and wash in a Buchner funnel with one 200-mLaliquot of toluene, followed by two 200-mL aliquots of methanol.Transfer to a beaker and add 300 mL of 50:50 methanol:water, pH 5.5 withHCI. Suspend and let sit at room temperature for 1 hour. Filter ontoBuchner funnel and wash phase with methanol and acetone. Transfer to the1000-mL round-bottomed flask and dry in oven overnight.

[0149] Next, endcap by adding 20 mL of trimethylchlorosilane, 6 mL ofhexamethyl-disilane, 350 mL of toluene, 10 mL of chloroform, and 25 mLof pyridine to the dried bonded phase, and bring to reflux for 6 hours.Cool the resulting mixture, transfer to a Buchner funnel, and wash withthree 200-mL aliquots of methanol, followed by three 200-mL aliquots ofacetone. Air dry for at least 0.5 hour, and then dry in the oven at 100°C. overnight.

[0150] Submit a sample for elemental analysis.

[0151] The bonded phase is now ready for column packing.

EXAMPLE 3 Dioctyl Silyl Phase—C-8X2

[0152] Repeat all of the steps for CN bonded phase, but replace3-cyanopropylmethyldichlorosilane with 100 mL of dioctyldichlorosilane.

EXAMPLE 4 Acid Wash Treatment

[0153] The procedures of Example 1 are repeated but the silica is washedwith 500 mL of 100 mM HCI and then water prior to drying. The product iswashed with 500 mL of 100 mM HCI after cooling and prior to the methanolwash.

EXAMPLE 5

[0154] The product of Example 1 is coated with 100 mL of dichloromethanecontaining 1 gram of divinylbenzene and 10 mg of benzoylperoxide. Thedichloromethane is removed by rotary evaporation until the monomer iscoated onto the beads. While rotating very slowly, the temperature isincreased to 70° C. for 8 hours. The product is washed with methanol.

[0155] This procedure is repeated with the product of Example 4.

EXAMPLE 6

[0156] The procedure of Example 5 is repeated with stearyldivinylbenzene in place of divinylbenzene. This procedure is repeated with theproduct of Example 4.

EXAMPLE 7

[0157] Fifteen (15) grams of the nonporous silica particles, 50 mL of2,2,4-trimethylpentane, and 25 mL of vinyltrichlorosilane are refluxedfor 2 hours. The modified silica is then washed several times with both2,2,4-trimethylpentane and acetone and dried at 80° C.

[0158] Five (5) grams of the vinyl-coated silica particles prepared asdescribed above are placed in a round bottom flask. Twenty-five mL ofacetonitrile containing 2 g of a vinyl monomer (divinylbenzene, styrene,acrylonitrile, acrylic acid, butyl methacrylate, or 2-hydroxymethacrylate) are added and the mixture well dispersed. Twenty-five mLof acetonitrile containing 0.2 g of dibenzoyl peroxide is added, and themixture is refluxed for 2 hours.

[0159] The products are extracted with acetonitrile and then acetone toremove unreacted monomers and oligomers from the particle.

[0160] In the case of the acrylic acid-modified silica, extractions withwater are also carried out.

[0161] The packing materials are dried at 80° C. prior to packing.

EXAMPLE 8 Standard Procedure for Testing the Performance of SeparationMedia

[0162] Separation particles are packed in an HPLC column and tested fortheir ability to separate a standard DNA mixture. The standard mixtureis a pUC18 DNA-HaeIII digest (Sigma-Aldrich, D6293) which contains 11fragments having 11, 18, 80, 102, 174, 257, 267, 298, 434, 458, and 587base pairs, respectively. The standard is diluted with water and fiveμL, containing a total mass of DNA of 0.25 μg, is injected.

[0163] Depending on the packing volume and packing polarity, theprocedure requires selection of the driving solvent concentration, pH,and temperature. The separation conditions are adjusted so that theretention time of the 257, 267 peaks is about 6 to 10 minutes. Any oneof the following solvents can be used: methanol, ethanol, 2-propanol,1-propanol, tetrahydrofuran (THF), or acetonitrile. A counter ion agentis selected from trialkylamine acetate, trialkylamine carbonate,trialkylamine phosphate, or any other type of cation that can form amatched ion with the polynucleotide anion.

[0164] As an example of this procedure, FIG. 4 shows the high resolutionof the standard DNA mixture using octadecyl modified, nonporouspoly(ethylvinylbenzene-divinylbenzene) beads. The separation wasconducted under the following conditions: Eluent A: 0.1 M TEAA, pH 7.0;Eluent B: 0.1 M TEAA, 25% acetonitrile; Gradient: Time (min) % A % B 0.065 35 3.0 45 55 10.0 35 65 13.0 35 65 14.0 0 100 15.5 0 100 16.5 65 35

[0165] The flow rate was 0.75 mL/min, detection UV at 260 nm, columntemp. 50° C. The pH was 7.0.

[0166] As another example of this procedure using the same separationconditions as in FIG. 4, FIG. 5 is a high resolution separation of thestandard DNA mixture on a column containing nonporous 2.1 micron beadsof underivatized poly(styrene-divinylbenzene).

EXAMPLE 9

[0167] This example demonstrates the high resolution separation of DNArestriction fragments using octadecyl modified, nonporous silica reversephase material, as described in Example 1. The experiment is conductedunder the following conditions: Column: 50×4.6 mm i.d. Mobile phase: 0.1M TEAA, pH 7.0. Gradient: 8.75-11.25% acetonitrile in 2 minutes,followed by 11.25-14.25% acetonitrile in 10 minutes, 14.5-15.25%acetonitrile in 4 minutes, and by 15.25-16.25% acetonitrile in 4minutes. Flow rate 1 mL/min. Column temperature: 50° C. Detection: UV at254 nm. Sample: Mixture of 0.75 μg pBR322 DNA-HaeIII restriction digestand 0.65 μg Φ×174 DNA-Hinc II restriction digest.

[0168] A high resolution separation is obtained by optimizing theconcentration of triethylammonium acetate (TEAA), shape of the gradientcurve, column temperature, and flow rate. The resolution of peaks iscontinuously enhanced in going from 25 mM to at least 125 mM of TEAA.The gradient is optimized by decreasing the steepness of the gradientcurve with increasing fragment lengths of DNA molecules. The bestseparations of double-stranded DNA molecules are accomplished at about30° C. to 50° C. Denaturation of DNA at higher than about 50° C.prevents utilization of higher column temperatures for double-strandedDNA fragments, although single-stranded DNA separations can be performedat temperatures up to 80° C. and higher.

EXAMPLE 10

[0169] If the gradient delay volume is minimized, the separation of PCRproducts and hybrid DNA derived from various sources of DNA, includingliving and dead organisms (animal and plant), as well as parts of suchorganisms (e.g., blood cells, biopsies, sperm, etc.) on octadecylmodified, nonporous poly-(ethylvinylbenzene-divinylbenzene) coated beadscan be achieved with run times under 2 minutes.

[0170] The analysis of PCR products and hybrid DNA usually requires onlyseparation and detection of one or two species of known length. Becauseof this, the resolution requirements are considerably less severe thanfor separations of DNA restriction fragments. Such less stringentresolution requirements allow the utilization of steep gradients and,consequently, lead to still shorter run times. The recovery rate for aDNA fragment containing 404 base pairs is about 97.5%.

[0171] Unlike capillary electrophoresis (CE), PCR samples do not have tobe desalted prior to analysis by MIPC. This represents a decisiveadvantage of MIPC over CE. With MIPC, it is thus possible to achieve afully automated analysis of PCR samples if an automatic autosampler isutilized. Moreover, since the volume of sample injection is known, incontrast to CE, quantitation over several orders of magnitude can beachieved without the need for an internal standard, hence allowing thequantitation of gene expression, as well as the determination of virustiters in tissues and body fluids. A fully automated version of themethod of the invention can be used to discriminate (distinguish) normalfrom mutated genes, as well as to detect oncogenes, bacterial and viralgenome polynucleotides (hepatitis C virus, HIV, tuberculosis) fordiagnostic purposes. Moreover, adjustment of column temperature allowsone to moderate the stringency of hybridization reactions or to separateheteroduplex from homoduplex DNA species.

[0172] The suitability of the polymer-coated beads of the invention forclinical use is described under the following conditions: Column: 50×4.6mm i.d. Mobile phase: 0.1 M TEAA, pH 7.0. Gradient: 11.25-13.75%acetonitrile in 1 minute, followed by 22.5% acetonitrile for 6 seconds,and 11.25% acetonitrile for 54 seconds., Flow rate: 3 mL/min. Columntemperature: 50° C. Detection: UV at 256 nm. Sample: 20 μl of a PCRsample. In the separation, the following elution order is obtained:1=unspecific PCR product, 2=PCR product having 120 base pairs, 3=PCRproduct having 132 base pairs, and 4=PCR product having 167 base pairs.

[0173] PCR methods and processes are described by R. K. Saiki et al. inScience, 23):1350-1354 (1985) and K. B. Mullis in U.S. Pat. No.4,863,202. These references are incorporated herein by reference for amore complete description of methods and processes for obtaining PCRsamples which can be separated using the method of the presentinvention.

[0174] The repetitive analysis of PCR products using the method of theinvention is highly reproducible under the described analyticalconditions. The results are not in any way influenced by the precedinginjection. The present method is highly suitable for routine use underreal conditions in clinical laboratories.

EXAMPLE 11

[0175] The following describes a separation of single-stranded DNA. Asilica-C18 column, as described in Example 1, 1.5 micron, 30×4.6 mmi.d., is used with a linear gradient of 2.5-12.5% acetonitrile in 0.1 Mtriethylammonium acetate in 40 minutes at 1 mL/min and 40° C. A mixtureof p(dC)12-18 and p(dT)12-18 oligonucleotides is separated, with thefirst mixture eluting between 5 and 15 minutes, and the second mixtureeluting between 15 and 30 minutes.

EXAMPLE 12 Sorption Enthalpy Measurements

[0176] Four fragments (174 base pair, 257 base pair, 267 base pair, and298 base pair, found in 5 μl pUC18 DNA-HaeIII digest, 0.04 μg DNA/μl) ofa DNA digest are separated under isocratical conditions at differenttemperatures using C-18 alkylated poly(styrene-divinylbenzene) polymerbeads. Conditions used for the separation are: Eluent: 0.1 Mtriethylammonium acetate, 14.25% (v/v) acetonitrile at 0.75 mL/min,detection at 250 nm UV, temperatures at 35, 40, 45, 50, 55, and 60° C.,respectively. A plot of In k versus 1/T (FIG. 6) shows that theretention factor k is increasing with increasing temperature. Thisindicates that the retention mechanism is based on an endothermicprocess (ΔH_(sorp)>0).

[0177] The same experiments on non-alkylatedpoly(styrene-divinylbenzene) beads (FIG. 7) give a negative slope for aplot of In k versus 1/T, although the plot is slightly curved.

[0178] The same experiments on alkylated poly(styrene-divinylbenzene)beads but the acetonitrile solvent is substituted with methanol (FIG. 8)gives a plot In k versus 1/T shows the retention factor k is decreasingwith increasing temperature. This indicates the retention mechanism isbased on an exothermic process (ΔH_(sorp)<0). Replacing the alkylatedand non-alkylated polymer beads with silica beads having a coating ofalkylated poly(styrene-divinylbenzene) and non-alkylated alkylatedpoly(styrene-divinylbenzene) will give the same results.

EXAMPLE 13 Separations with Alkylated poly(styrene-divinylbenzene) Beads

[0179] Mobile phase components are chosen to match the desorptionability of the elution solvent in the mobile phase to the attractionproperties of the bead to the DNA-counter ion complex. As the polarityof the bead decreases, a stronger (more organic) or higher concentrationof solvent will be required. Weaker organic solvents such as methanolare generally required at higher concentrations than stronger organicsolvents such as acetonitrile.

[0180]FIG. 9 shows the high resolution separation of DNA restrictionfragments using octadecyl modified, nonporouspoly(ethylvinylbenzene-divinylbenzene) beads. The experiment wasconducted under the following conditions. Column: 50×4.6 mm i.d.; mobilephase 0.1 M triethylammonium acetate (TEAA), pH 7.2; gradient: 33-55%acetonitrile in 3 min, 55-66% acetonitrile in 7 min, 65% acetonitrilefor 2.5 min; 65-100% acetonitrile in 1 min; and 100-35% acetonitrile in1.5 min. The flow rate was 0.75 mL/min, detection UV at 260 nm, columntemp. 51° C. The sample was 5 μl (=0.2 μg pUC18 Hea III digest).

[0181] Repeating the above procedure replacing the acetonitrile with50.0% methanol in 0.1 M TEAA gives the separation shown in FIG. 10.

[0182] Repeating the above procedure replacing the acetonitrile with25.0% ethanol in 0.1 M TEAA gives the separation shown in FIG. 11.

[0183] Repeating the above procedure replacing the acetonitrile with 25%vodka (Stolichnaya, 100 proof) in 0.1 M TEAA gives the separation shownin FIG. 12.

[0184] The separation shown in FIG. 13 was obtained using octadecylmodified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads asfollows: Column: 50×4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;gradient: 12-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 3 min,18-22% B in 7 min, 22% B for 2.5 min; 22-100% B in 1 min; and 100-1 2% Bin 1.5 min. The flow rate was 0.75 mL/min, detection UV at 260 nm, andcolumn temp. 51° C. The sample was 5 μl (=0.2 μg pUC18 DNA-HaeIIIdigest).

[0185] The separation shown in FIG. 14 was obtained using octadecylmodified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads asfollows: Column: 50×4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;gradient: 15-18% 0.1 M TEAA and 25.0% 1-propanol (Eluent B) in 2 min,18-21% B in 8 min, 21% B for 2.5 min; 21-100% B in 1 min; and 100-1 5% Bin 1.5 min. The flow rate was 0.75 mL/min, detection UV at 260 nm, andcolumn temp. 510° C. The sample was 5 μl (=0.2 μg pUC18 DNA-HaeIIIdigest).

[0186] The separation shown in FIG. 15 was obtained using octadecylmodified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads asfollows: Column: 50×4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;gradient: 35-55% 0.1 M TEAA and 10.0% 2-propanol (Eluent B) in 3 min,55-65% B in 10 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-35% Bin 1.5 min. The flow rate was 0.75 mL/min, detection UV at 260 nm, andcolumn temp. 51° C. The sample was 5 μl (=0.2 μg pUC18 DNA-HaeIIIdigest).

[0187] The separation shown in FIG. 16 was obtained using octadecylmodified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads asfollows: Column: 50×4.6 mm i.d.; mobile phase 0.05 M TEA₂HPO₄, pH 7.3;gradient: 35-55% 0.05 M TEA₂HPO₄and 10.0% 2-propanol (Eluent B) in 3min, 55-65% B in 7 min, 65% B for 2.5 min; 65-100% B in 1 min; and100-65% B in 1.5 min. The flow rate was 0.75 mL/min, detection UV at 260nm, and column temp. 51° C. The sample was 5 μl (=0.2 μg pUC18DNA-HaeIII digest).

[0188] The separation shown in FIG. 17 was obtained using octadecylmodified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads asfollows: Column: 50×4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;gradient: 6-9% 0.1 M TEAA and 25.0% THF (Eluent B) in 3 min, 9-11% B in7 min, 11% B for 2.5 min; 11-100% B in 1 min; and 100-6% B in 1.5 min.The flow rate was 0.75 mL/min, detection UV at 260 nm, and column temp.51° C. The sample was 5 μl (=0.2 μg pUC18 DNA-HaeIII digest).

EXAMPLE 14 Isocratic/Gradient Separation of dsDNA

[0189] The following is an isocratic/gradient separation of dsDNA on apolystyrene coated silica base material. Isocratic separations have notbeen performed in DNA separations because of the large differences inthe selectivity of DNA/alkylammonium ion pair for beads. However, byusing a combination of gradient and isocratic elution conditions, theresolving power of a system can be enhanced for a particular size rangeof DNA. For example, the range of 250-300 base pairs can be targeted byusing a mobile phase of 0.1 M TEAA, and 14.25% acetonitrile at 0.75mL/min at 40° C. on a 50.×4.6 mm crosslinked polystyrene coated silicareverse phase column, 2.0 micron. The pUC18 DNA-HaeIII digest wasinjected under isocratic conditions and 257, 267 and 298 base pairs DNAeluted completely resolved. Then larger fragments were removed from thecolumn with 0.1M TEAA/25% acetonitrile at 9 minutes. FIG. 18 shows aseparation using the same elution conditions but performed on apoly(styrene-divinylbenzene) polymer based column. In other examples,there might be an initial isocratic step (to condition the column), thena gradient step (to remove or target the first group of DNA at aparticular size), then an isocratic step (to separate the targetmaterial of a different size range) and finally a gradient step to cleanthe column.

EXAMPLE 15 Preparation of a Silica Monolith

[0190] All the gel samples are prepared by hydrolyzingtetramethoxysilane (TMOS) with 0.01M aqueous solution of acetic acid inthe presence of poly(ethylene oxide)(PEO), with an average molecularweight of 10,000. The PEO containing system is chosen because it givesrelatively high macropore volume and exhibits less sensitive dependenceof pore size on compositional parameters. The constituents are mixedtogether in an ice cooled container and stirred vigorously for 5 min,while the hydrolysis gradually proceeds and the heterophase mixture ishomogenized due to the liberation of alcohol. The solution is thendegassed under ultrasonic radiation, and is poured into plasticcylinders with inner diameter of 10 mm. The solution is then kept atconstant temperatures for gelation and aging, in a tightly closedcontainer. The well-aged gels are subsequently immersed in a 0.2Maqueous ammonia solution for 10 days to exchange the solvent phase, thendried at 60° C. for 3 days and finally heat treated at 600° C. for 2 h.The chemical modification with octadecyl (hereafter denoted as C18) andmethyl ligands of the inner surface of the gel samples thus prepared iscarried out according to the method reported below.

[0191] The base silica has a nominal through pore diameter of 5 μm and asubstrate pore size of 110 angstroms. The following synthesis procedureis employed: the silica gel is first dried at 150° C. for 2 hours, thendried under reduced pressure (30 mm Hg) for an additional 2 hours. Thederivatization reactions are performed with 10 g of silica gel and 8.5micromol/m² equivalents of silane compound in pyridine and toluene.Trichlorooctadecylsilane is used for the preparation of trifunctionalphases, dichloromethyloctadecylsilane for difunctional phases, andmoncholorodimethyloctadecylane for monofunctional phases. The mixture ofsilica gel and silane are refluxed for 5 hours at 100° C. in toluene.After cooling, the packings are dispersed in chloroform, filtered, andwashed several lines with chloroform. The final packings are dried at80° C. for 8 hours. Endcapping is performed using 8.5 micromol/m²equivalents of trimethylchlorosilane and hexamethyldisilazane underrefluxing pyridine and toluene for 5 hours. The liquid chromatographicmeasurements are carried out for the gel rods coherently clad withthermoshrinking PTFE resin and equipped with suitable connectiondevices.

EXAMPLE 16 Acid Wash Treatment to Remove Multivalent Metal CationContaminants

[0192] The non-polar, derivatized silica monolith column is washed byflowing tetrahydrofuran through the column at a flow rate of 2 mL perminute for 10 minutes followed by flowing methanol through the column at2 mL per minute for 10 minutes. The non-polar monolith column is washedfurther by flowing a mixture containing 100 mL of tetrahydrofuran and100 mL of concentrated hydrochloric acid through the column at 10 mL perminute for 20 minutes. Following this acid treatment, the monolithcolumn is washed by flowing tetrahydrofuran/water (1:1) through thecolumn at 2 mL per minute until neutral (pH 7).

EXAMPLE 17 Determination of the Mutation Separation Factor

[0193] The Mutation Separation Factor (MSF) is determined by thefollowing equation:

MSF=(area peak 2−area peak 1)/area peak 1

[0194] where area peak 1 is the area of the peak measured after DMIPCanalysis of wild type and area peak 2 is the total area of the peak orpeaks measured after DMIPC analysis of a hybridized mixture containing aputative mutation, with the hereinabove correction factors taken intoconsideration, and where the peak heights have been normalized to thewild type peak height. Separation particles are packed in an HPLC columnand tested for their ability to separate a standard hybridized mixturecontaining a wild type 100 bp Lambda DNA fragment and the corresponding100 bp fragment containing an A to C mutation at position 51.

[0195] Depending on the packing volume and packing polarity, theprocedure requires selection of the driving solvent concentration, pH,and temperature. Any one of the following solvents can be used:acetonitrile, tetrahydrofuran, methanol, ethanol, or propanol. Any oneof the following counterion agents can be used: trialkylamine acetate,trialkylamine carbonate, and trialkylamine phosphate.

[0196] As an example of the determination of the Mutation SeparationFactor, FIG. 24 shows the resolution of the separation of the hybridizedDNA mixture.

[0197] The PCR conditions used with each of the primers are described inthe table below. All the components were combined and vortexed to ensuregood mixing, and centrifuged. Aliquots were then distributed into PCRtubes as shown in the following table: COMPONENT VOLUME Pfu 10X Buffer(Cat. No. 5 μL 600153-82, Stratagene, Inc., La Jolla, CA) 1100 μM dNTPMix 4 μL Primer 1 7.5 μL (forward) Primer 2 8.5 μL (reverse) H₂O 19.5 μLLambda DNA Template 5 μL PFUTurbo ™ 0.5 μL (600250, Stratagene)

[0198] The PCR tubes were placed into a thermocycler (PTC-100Programmable Thermal Controller from MJ Research, Inc., Watertown,Mass.) and the temperature cycling program was initiated. The cyclingprogram parameters are shown in the table below: STEP TEMPERATURE TIME 194° C. 2 minutes 2 94° C. 1 minute 3 58° C. 1 minute 4 72° C. 1 minute 5Go to Step 2, 34X 6 72° C. 10 minutes 7 End

[0199] The DMIPC conditions used for the mutation detection separationsare shown below:

[0200] Eluent A: 0.1 M TEAA; Eluent B: 0.1 M TEAA, 25% Acetonitrile;Flow rate: 0.90 mL/min; Gradient: Time (min) % A % B 0.0 50.0 50.0 0.145.0 55.0 4.6 36.0 64.0 4.7 0.0 100.0 5.2 0.0 100.0 5.3 50.0 50.0 7.850.0 50.0

[0201] The Lambda sequence has been published by O'Conner et at. inBiophys. J. 74:A285 (1998) and by Garner, et al., at the MutationDetection 97 4th International Workshop, Human Genome Organization, May29-Jun. 2, 1997, Brno, Czech Republic, Poster no. 29. The 100 bp Lambdafragment sequence (base positions 32011-32110) was used as a standard(available from FMC Corp. available from FMC Corp. BioProducts,Rockland, Me.). The mutation was at position 32061. The chart belowlists the primers used: Primers Forward Primer:5′-GGATAATGTCCGGTGTCATG-3′ Reverse Primer: 3′-GGACACAGTCAAGACTGCTA-5′

[0202]FIG. 23 is a chromatogram of the wild type strand analyzed underthe above conditions. The peak appearing has a retention time of 4.78minutes and an area of 98621.

[0203]FIG. 24 is the Lambda mutation analyzed in identical conditions asFIG. 23 above. Two peaks are apparent in this chromatogram, withretention times of 4.32 and 4.68 minutes and a total area of 151246.

[0204] The Mutation Separation Factor is calculated by applying thesevarious peak areas to the above MSF equation. Thus, using the definitionstated hereinabove, MSF=(area peak 2−area peak 1)/area peak 1, the MSFwould be (151246−98621)/98621, or 0.533.

EXAMPLE 18 Effect of Multivalent Cation Decontamination Measures onSample Resolution by DMIPC

[0205] The separation shown in FIG. 20 was obtained using a WAVE™ DNAFragment Analysis System (Transgenomic, Inc., San Jose, Calif.) underthe following conditions: Column: 50×4.6 mm i.d. containing alkylatedpoly(styrene-divinylbenzene) beads (DNASep®, Transgenomic, Inc.); mobilephase 0.1 M TEAA (1 M concentrate available from Transgenomic, Inc.)(Eluent A), pH 7.3; gradient: 50-53% 0.1 M TEAA and 25.0% acetonitrile(Eluent B) in 0.5 min; 53-60% B in 7 min; 60-100% B in 1.5 min; 100-50%B in 1 min; 50% B for 2 min. The flow rate was 0.9 mL/min, UV detectionwas at 254 nm, and the column temperature was 56° C. The sample was 2 μL(=0.2 μg DNA, DYS271 209 bp mutation standard with an A to G mutation atposition 168).

[0206]FIG. 21 is the same separation as performed in FIG. 20, but afterchanging the guard cartridge (20×4.0 mm, chelating cartridge, part no.530012 from Transgenomic, Inc.) and replacing the pump-valve filter(Part no. 638-1423, Transgenomic, Inc.). The guard cartridge haddimensions of 10×3.2 mm, containing iminodiacetate chelating resin of2.5 mequiv/g capacity and 10 μm particle size, and was positioneddirectly in front of the injection valve.

[0207]FIG. 22 is the same separation as performed in FIG. 21, but afterflushing the column for 45 minutes with 0.1M TEAA, 25% acetonitrile, and32 mM EDTA, at 75° C.

EXAMPLE 19 Hybridization of Mutant and Wild Type DNA Fragments

[0208] A mixture of two homoduplexes and two heteroduplexes was producedby a hybridization process. In this process, a DYS271 209 bp mutationstandard containing a mixture of the homozygous mutant DNA fragment(with an A to G mutation at position 168) combined with thecorresponding wild type fragment in an approximately 1:1 ratio (themixture is available as a Mutation Standard from Transgenomic, Inc., SanJose, Calif.; the mutation is described by Seielstad et al., Human Mol.Genet. 3:2159 (1994)) was heated at 95° C. for 3-5 minutes then cooledto 25° C. over 45 minutes. The hybridization process is shownschematically in FIG. 19.

[0209] While the foregoing has presented specific embodiments of thepresent invention, it is to be understood that these embodiments havebeen presented by way of example only. It is expected that others willperceive and practice variations which, though differing from theforegoing, do not depart from the spirit and scope of the invention asdescribed and claimed herein.

The invention claimed is:
 1. A method for separating a mixture of polynucleotides, comprising applying a mixture of polynucleotides having up to 1500 base pairs to a separation medium, the separation surfaces of said medium coated with a hydrocarbon or non-polar hydrocarbon substituted polymer, or having substantially all polar groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group, wherein said surfaces are non-polar; and eluting said mixture of polynucleotides.
 2. A method of claim 1 wherein said medium is characterized by having a DNA Separation Factor of at least 0.05.
 3. A method of claim 1 wherein said medium is characterized by having a Mutation Separation Factor of at least 0.1.
 4. A method of claim 1 including eluting said mixture with a mobile phase comprising a counterion agent and an organic solvent, wherein said organic solvent is water soluble.
 5. A method of claim 4 , wherein said solvent is selected from the group consisting of alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof.
 6. A method of claim 4 wherein said counterion agent is selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower alkyl trialkyammonium salt, quaternary ammonium salt, and mixtures of one or more thereof.
 7. A method of claim 4 wherein said counterion agent is selected from the group consisting of octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, a triethylammonium hexafluoroisopropyl alcohol, and mixtures of one or more thereof.
 8. A method of claim 4 wherein said counterion agent includes an anion, said anion is selected from the group comprising acetate, carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide.
 9. A method of claim 1 wherein said separation is by Matched Ion Polynucleotide Chromatography.
 10. The medium of claim 1 wherein said medium is subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants from said surface.
 11. A method of claim 1 wherein said medium comprises beads having an average diameter of 0.5 to 100 microns, said beads comprising nonporous particles.
 12. A method of claim 4 wherein said beads are characterized by having a Mutation Separation Factor of at least 0.1.
 13. A method of claim 11 wherein said beads are characterized by having a DNA Separation Factor of at least 0.05.
 14. A method of claim 11 wherein said nonporous particles are a member selected from the group consisting of silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharide, and diatomaceous earth.
 15. A method of claim 11 wherein said nonporous particles are silica.
 16. A method of claim 15 wherein said nonporous beads are substantially free from unreacted silanol groups.
 17. A method of claim 11 wherein said separation is made by capillary electrochromatography.
 18. A method of claim 11 wherein said separation is made by thin layer chromatography or high-speed thin layer chromatography.
 19. A method of claim 1 wherein the non-polar surfaces are the surfaces of interstitial spaces of a silica gel monolith.
 20. A method of claim 19 wherein said monolith has been subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants.
 21. A method of claim 19 wherein said monolith is characterized by having a DNA Separation Factor of at least 0.05.
 22. A method of claim 19 wherein said monolith is characterized by having a Mutation Separation Factor of at least 0.1.
 23. A method of claim 19 wherein said surfaces are substantially free form unreacted silanol groups.
 24. A method of claim 19 wherein said monolith has substantially all separation surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group.
 25. A method of claim 19 including eluting said mixture with a mobile phase comprising a counterion agent and an organic solvent, wherein said organic solvent is water soluble.
 26. A method of claim 19 wherein said separation is by Matched Ion Polynucleotide Chromatography.
 27. A bead comprising a nonporous particle coated with a polymer, wherein said bead has an average diameter of 0.5 to 100 microns and wherein said bead is characterized by having a Mutation Separation Factor of at least 0.1.
 28. A bead of claim 27 wherein said bead is characterized by having a DNA Separation Factor of 0.05.
 29. A bead of claim 27 wherein said bead is subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants.
 30. A bead of claim 27 wherein said nonporous particle is selected from the group consisting of silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharides, and diatomaceous earth.
 31. A bead of claim 27 wherein said nonporous particle is nonporous silica reacted to make a reverse phase material.
 32. A bead of claim 31 wherein said nonporous bead is substantially free from unreacted silanol groups.
 33. A bead comprising a nonporous particle having substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group, wherein said bead has an average diameter of 0.5 to 100 microns and wherein said bead is characterized by having a Mutation Separation Factor of at least 0.1 using Matched Ion Polynucleotide Chromatography.
 34. A method for treating the bead of claim 27 in order to improve the resolution of polynucleotides separated using said bead, comprising contacting a solution containing a multivalent cation binding agent with said bead, wherein said solution has a temperature of about 50° C. to 90° C.
 35. A method for storing the bead of claim 27 in order to improve the resolution of polynucleotides separated using said bead, comprising contacting a solution containing a multivalent cation binding agent with said beads prior to storing said bead.
 36. A silica gel monolith having non-polar interstitial separation surfaces wherein said monolith has been subjected to an acid wash treatment in order to substantially remove multivalent cation contaminants from said surfaces.
 37. A silica gel monolith of claim 36 having substantially all separation-surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group.
 38. A monolith of claim 36 characterized by having a DNA Separation Factor of at least 0.05.
 39. A monolith of claim 36 characterized by having a Mutation Separation Factor of at least 0.1.
 40. A method for treating the monolith of claim 36 in order to improve the resolution of polynucleotides separated using said monolith, comprising contacting a solution containing a multivalent cation binding agent with said surfaces, wherein said solution has a temperature of about 50° C. to 90° C.
 41. A method for storing the monolith of claim 36 in order to improve the resolution of polynucleotides separated using said monolith, comprising contacting a solution containing a multivalent cation binding agent with the separation surfaces of said monolith prior to storing said monolith. 